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Full text of "Assessment of ethanol toxicity in the motor system of the chick and the septohippocampal system of the rat and the involvement of neurotrophic factors"

ASSESSMENT OF ETHANOL TOXICITY IN THE MOTOR SYSTEM OF THE CHICK 

AND THE SEPTOHIPPOCAMPAL SYSTEM OF THE RAT AND THE 

INVOLVEMENT OF NEUROTROPHIC FACTORS 



By 

DOUGLAS M. BRADLEY 



A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE 

UNIVERSITY OF FLORIDA IN PARTIAL FULHLLMENT OF THE REQUIREMENTS 

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 
1998 



ACKNOWLEDGMENTS 
I would like to acknowledge the help of many people who made completion of this 
dissertation and doctoral research possible. First and foremost, I would like to thank my 
wife, Korey, who provided valuable mental support and editing and presentation advice 
through the years. I also want to thank my advisor, Marieta Heaton, who provided the 
means for completing this research and provided excellent guidance. My committee, Drs. 
MacLennan, Shiverick, Streit, and Walker, really helped to make this research better. My 
father. Dr. Exiwin L. Bradley, provided valuable advice on statistics. I want to thank my 
family, for understanding the time required to complete this degree. I also want to thank 
the National Science Foundation and NIAAA, for supporting me financially through 
graduate school. The Department of Neuroscience provided an excellent facility for 
conducting this research. I also want to thank all of the people in our laboratory who 
helped in a variety of technical and supportive ways throughout the years. Blaine Moore, 
has been a friend in addition to giving helpful advice on scientific matters. Francesca 
Beaman, Steve Farnworth, Kara Kidd, Nancy MacLennan, David Melman, Jean Mitchell, 
Micheal Paiva, and Leon Williams gave wonderful technical assistance. I also wish to 
apologize to anyone who is unintentionally omitted from this list. 



TABLE OF CONTENTS 

gage 

ACKNOWLEDGMENTS u 

LIST OF TABLES v 

LIST OF FIGURES vi 

ABSTRACT i^^ 

CHAPTERS 

1 INTRODUCTION 1 

Fetal Alcohol Syndrome Background 1 

Chick Embryo 3 

Motor System and Ethanol 5 

Motor System and NTFs 5 

i Neuroprotection 7 

Rat Model of FAS 9 

Rat Septohippocampal System 10 

Neurotrophic Factors • • 1 1 

Gene Deletion Studies 12 

NTF Ontogeny in the Hippocampus 14 

Neurotrophins and Ethanol 15 

Hypotheses 16 

2 CHARACTERIZATION OF MOTONEURON SURVIVAL AND CELL 
DEATH FOLLOWING ETHANOL EXPOSURE AND CURARE 
ADMINISTRATION, AND AFTER THE PERIOD FOR 
NATURALLY OCCURRING CELL DEATH 18 

Summary 18 

Introduction 18 

Methods 21 

Results 29 

Discussion 38 

3 CHARACTERIZATION OF MOTONEURON SURVIVAL FOLLOWING 
ETHANOL EXPOSURE AND CONCURRENT TREATMENT 
WITH EXOGENOUS GDNF OR BDNF IN THE EMBRYONIC 
CHICK SPINAL CORD 49 

Summary 49 

I ntroduction 50 

Materials and Methods 55 



111 



Results 58 

Discussion 67 

4 CHARACTERIZATION OF THE NEUROTROPHIN AND 

NEUROTROPHIN RECEPTOR GENE EXPRESSION IN THE 
HIPPOCAMPUS FOLLOWING CHRONIC TREATMENT AND 
EARLY POSTNATAL ETHANOL TREATMENT IN THE RAT 75 

Summary 75 

Introduction 76 

Materials and Methods 83 

Results 88 

Discussion 133 

5 CONCLUSIONS AND IMPLICATIONS 146 

Animal Models 146 

Methods 147 

Hypotheses and Results 150 

Conclusions 154 

REFERENCES 158 

BIOGRAPHICAL SKETCH 178 






'5 



IV 



LIST OF TABLES 
Table page 

2- L Cell size and spinal cord length 35 

2-2. Neurotrophic activity of crude muscle extract 37 

3-1. Motoneuron Size and Spinal Cord Length 59 



LIST OF HGURES 

Figure pa ge 

2-1. Number of motoneurons in lumbar spinal cord at E12 following treatment 31 

from E4toEll. 

2-2. Photomicrographs of coronal sections from the midlumbar region of E12 34 

spinal cords. 

2-3. Number of motoneurons in lumbar spinal cord at E16 following ethanol 36 

treatment from ElO to E15. 

3-1. Number of motoneurons in the later motor column of the lumbar spinal cord 61 

at E16. 

3-2. Interaction between ethanol and neurotrophic factors 62 

3-3. Photomicrographs of coronal sections from the midlumbar region of E16 64 

spinal cords. 

3-4. High magnification photomicrographs from the midlumbar section of E16 66 

spinal cords. 

4-1. Brain weight at P21 of female and male animals following prenatal ethanol 90 

exposure. 

4-2. Weight gain during postnatal ethanol exposure in male animals 92 

4-3. Gross morphological measurements following EPET in male animals at P21 94 

4-4. Brain weight and Brain weight to body weight ratio of EPET male animals 95 

atP21. 

4-5. Weight gain during postnatal ethanol exposure in female animals 96 

4-6. Brain weight and brain weight to body weight ratio in EPET female animals 98 

atP21. 

4-7. Phosphorimaging view of BDNF Northern blots composed of the 100 

hippocampal region from P21 rat brains exposed to ethanol prenatally. 

4-8. Relative BDNF 4.4 kb transcript expression in rat hippocampus at P21 101 

following prenatal exposure to ethanol. 

4-9. Relative BDNF 1.7 kb transcript gene expression following prenatal 102 

exposure in P21 rats. 



VI 



4-10. Phosphorimaging view of NT -3 Northern blots composed of the 104 

hippocampal region from P21 rat brains exposed to ethanol prenatally. 

4-11. Relative NT-3 gene expression following prenatal ethanol exposure in P21 105 

rats. 

4-12. Relative trkB active receptor gene expression following prenatal ethanol 106 

exposure in P21 rats. 

4-13. Relative trkB truncated transcript gene expression following prenatal 107 

exposure in P21 rats. 

4- 14. Phosphorimaging view of trkC Northern blots composed of the 109 

hippocampal region from P21 rat brains exposed to ethanol prenatally. 

4-15. Relative trkC 14 kb transcript gene expression at P21 in rats exposed to 110 

ethanol prenatally. 

4- 16. Relative trkC 4.7 kb truncated transcript gene expression following HI 

prenatal exposure in P21 rats. 

4- 17. Relative trkC 3.9 kb transcript gene expression following prenatal 112 

exposure in P21 rats. 

4-18. Phosphorimaging view of cyclophilin Northern blots composed of the 115 

hippocampal region from P21 rat brains exposed to ethanol prenatally. 

4-19. Phosphorimaging view of BDNF Northern blots from postnatally 118 

exposed P21 rats. 

4-20. Relative BDNF 4.4 kb transcript gene expression following postnatal 119 

exposure in P21 rats. 

4-21. Relative BDNF 1.7 kb transcript gene expression following postnatal 120 

exposure in P21 rats. 

4-22. Phosphorimaging view of NT-3 Northern blots from postnatally exposed 122 

P21 rats. 

4-23. Relative NT-3 1.5 kb gene expression following postnatal exposure in 123 

P21 rats. 

4-24. Relative trkB active receptor gene expression following postnatal 124 

exposure in P21 rats. 

4-25. Relative trkB truncated transcript gene expression following postnatal 125 

exposure in P21 rats. 

4-26. Phosphorimagmg view of trkC Northern blots from postnatally exposed 127 

P21 rats. 

4-27. Relative trkC 14 kb transcript gene expression in P21 rats following 128 

postnatal exposure 



Vll 



4-28. Relative trkC 4.7 kb truncated transcript gene expression in P21 rats 129 

following postnatal exposure. 

4-29. Relative trkC 3.9 kb truncated transcript gene expressionin P21 rats 130 

following postnatal exposure. 

4-30. Phosphorimaging view of cyclophilin Northern blots from postnatally 132 

exposed P21 rats. 



viu 



Abstract of Dissertation Presented to the Graduate School of the University of Horida in 
Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 

ASSESSMENT OF ETHANOL TOXICITY IN THE MOTOR SYSTEM OF THE CHICK 

AND THE SEPTOHIPPOCAMPAL SYSTEM OF THE RAT AND THE 

INVOLVEMENT OF NEUROTROPHIC FACTORS 

By 

Douglas M. Bradley 

August 1998 

Chairman: Douglas K. Anderson 
Major Department: Neuroscience 

The research described in this document was undertaken to further the 

understanding of the toxic effects that ethanol exerts on the developing nervous system. 

Fetal alcohol syndrome has been recognized as one of the leading environmentally-induced 

causes of mental retardation in the western world and continues to be a problem despite 

education and publicity concerning the dangers of ingesting ethanol-containing beverages 

during pregnancy. The doctoral research described attempted to ascertain some new 

properties of ethanol toxicity in the nervous system and to determine ways that these toxic 

effects could be modulated in living animals. Previous research from other laboratories has 

suggested that motoneurons of the spinal cord might be susceptible to ethanol's toxic 

effects. Our laboratory confirmed this finding by administering ethanol to developing chick 

embryos from embryonic day 4 (E4) to Ell and assessing the number of motoneurons 

present in the lumbar spinal cord. Specifically, a reduction in the number of motoneurons 

present in this population was observed. The present experiments found that embryonic 

administration of ethanol from ElO to E15 also results in a loss of motoneurons. Further, 

the neurotrophic activity of muscle from these animals is unchanged from that of control 

animals. Neuromuscular junction blocking agents, which prevent naturally occurring cell 

death of spinal cord motoneurons, have little effect in altering ethanol's toxic effects. 

Administration of glial cell line-derived neurotrophic factor acted to increase motoneuron 

ntimber following ethanol administration, but brain-derived neurotrophic factor did not. 

ix 



The hippocampus is an important structure of the brain thought to be involved with learning 
and memory. In a mammalian model of fetal alcohol syndrome, the gene expression of 
tyrosine receptor kinase C, a neurotrophic factor receptor in the brain, is reduced in the 
hippocampus of 21 -day-old male rats following prenatal ethanol exposure, but is 
unchanged in the brain of female rats. Appropriate background for understanding this 
research, as well as the implications of all of these results, is described in the resulting 
chapters. 



CHAPTER 1 
BACKGROUND INFORMATION 

Fetal Alcohol Syndrome Background 
In 1973, Jones and Smith (1973) first described a series of morphological and 
cognitive deficits in children and infants of alcoholic mothers which was later termed fetal 
alcohol syndrome (FAS). Since that time, much evidence has been gathered regarding the 
effects ethanol exerts in the developing nei-vous system (Barnes and Walker, 1981; Jones 
and Smith, 1973; Miller, 1986; Streissguth et al, 1991; West, 1986). FAS is diagnosed in 
1-2 out of every 1000 live births m the United States and is characterized by low birth 
weight, decreased memory and learning, hyperactivity, facial dysmorphia, and lowered IQ 
(Abel, 1995; Jones and Smith, 1973; Streissguth et al, 1991). Human FAS patients have 
been analyzed for neuropathology postmortem and this analysis has identified central 
nervous system (CNS) abnormalities which include disorders of laminae of the cerebral 
cortex, cerebellar abnormalities, a reduction of dendritic spines on cortical pyramidal cells, 
hippocampal malfonnation, and microcephaly (Clarren et al., 1978; Ferrer and Galofre, 
1987). Interpretation of these studies is complicated by the fact that most of these infants 
had related cardiovascular problems (Clarren et al., 1978). Abnormalities in humans can 
range from physical deficits that are easily distinguished (such as gross microencephaly) to 
microscopic changes (such as dendritic anomalies in neurons that survived alcohol 
exposure) that require finer analyses (Ferrer and Galofre, 1987). Motor dysfunction and 
other behavioral deficits, such as an impairment in sensory and motor functions, are 
associated with FAS (Streissguth et al., 1983). Additionally, children with FAS are 
deficient in habituation to redundant stimuli (Church and Gerkin, 1988). What is important 



to note is that as these patients have aged, the deficits have not lessened (Streissguth, 
1993). 

Specific neuronal populations known lo be affected by ethanol in animal models 
include the cerebellum (Cragg and Phillips, 1985; West, 1986), the septohippocampal 
system (Barnes and Walker, 1981), cerebral cortex (Miller, 1986), the substantia nigra 
(Shetty et al., 1993), chief sensory trigeminal nucleus (Miller and Muller, 1989), red 
nucleus (Zajac et al., 1989), infenor olivary nucleus (Napper and West, 1995), striatum 
(Heaton et al., 1996) and motoneurons of the spinal cord (Heaton and Bradley, 1995). 
Microscopic and molecular changes that have also been observed in animal models 
following ethanol exposure include decreased dendritic arborization (Davies and Smith, 
1981), delayed synaptogenesis (Leonard, 1987), decreased neurotransmitter synthesis 
(Rawat, 1977; Swanson et al., 1994), changes in connectivity (West et al, 1994), and cell 
loss (Barnes and Walker, 1981; Bauer-Moffet and Altman, 1975; West et al., 1986). 
These alterations following ethanol exposure in animals are important because they 
correlate to deficits observed in human FAS. That is, the neuronal region affected seems to 
relate to a specific deficiency common to human FAS patients. 

The mechanisms of ethanol toxicity in the CNS are not fully understood. Since 
ethanol can cross the blood brain barrier, it has the ability to directly affect the developing 
nervous system (West et al., 1994). Ethanol can interact with cellular membranes and 
proteins and reduce protein synthesis (Zajac and Abel, 1992). Additionally, ethanol has 
been implicated in producing hypoglycemia (Snyder et al., 1992; West et al., 1994), 
hypoxia (Mukherjee and Hodgen, 1982), and in increasing oxidative stress (Henderson et 
al., 1995). All of the above information suggest possible explanations for the toxic effects 
ethanol exerts on the developing nervous system. The current experiments were designed 
to test facets of the relationship between neurotrophic factors (NTFs) and ethanol. 
Important to these studies is the underlying hypothesis that NTFs are involved in FAS 
neuropathology. The NTF hypothesis for FAS proposes that ethanol exposure results in 



alterations in the synthesis, availabihty, delivery, and /or biological activity of normally 
occurring neurotrophic substances. Further, ethanol may alter the capacity of target 
neuronal populations to respond to NTFs in a normal fashion. Another important aspect of 
the NTF hypothesis is the idea that exogenous NTFs may afford some protection to 
ethanol-susceptible neuronal populations. The current studies sought to determine whether 
NTF synthesis, availability, and biological activity were affected by ethanol treatment and 
whether the addition of exogenous factors in vivo could prevent ethanol toxicity in a 
population known to be vulnerable to ethanol insult. To adequately examine these goals, 
two animal models were used: the chick embiyo and the developing rat. Each model has 
specific advantages that make it attractive for FAS research and each will be discussed in 
more detail below. All of the studies detailed in this document are related in that the 
examine ethanol toxicity as it relates to NTFs. Whether this relationship is in the effect that 
ethanol has on NTFs or neurotrophic support, or the effect that exogenous NTFs have on 
ethanol toxicity, the objective is consistent: to understand the manner in which these two 
types of molecules are related in producing deficits observed in animal models of FAS. 

Chick Embryo 
Ethanol affects chick embryo development in a manner similar to mammals. Chicks 
exposed to ethanol prenatally have been shown to exhibit reduced brain size, brain weight, 
DNA and protein synthesis (Pennington and Kalmus, 1987), and reduced neurotransmitter 
synthesis (Brodie and Vernadakis, 1990; Swanson et al., 1994). Neuronal populations 
affected by ethanol exposure in ovo in chick include the cerebellum (Quesada et al., 1990), 
cerebral cortex (Delphia et al., 1978), and motoneurons of the spinal cord (Heaton and 
Bradley, 1995). The cerebellum (Marcussen et al., 1994; Smith and Davies, 1990) and 
cerebral cortex (Miller, 1986) in mammals are also affected by developmental ethanol 
exposure. While chick motoneurons are susceptible to the toxic effects of ethanol both in 
culture (Dow and Riopelle, 1985; Heaton and Bradley, 1995) and in vivo (Heaton and 
Bradley, 1995), they have not been specifically quantified in mammals exposed to ethanol. 



4 

Some evidence does suggest that there are neuromuscular problems associated with human 
FAS. Specifically, children exposed to ethanol exhibit motor deficits (Streissguth et al., 
1983). More important to the current studies is the fact that ethanol can reduce motoneuron 
number when administered to chick embryos from embryonic day 4 (E4) to El 1 (Heaton 
and Bradley, 1995). We have hypothesized that this reduction may be dependent on 
naturally occurring cell death (NOCD) since this period (approximately E6-E9) occurred 
during the period of ethanol exposure utilized in that study (Pittman and Oppenheim, 
1978). During the period of NOCD, nearly half of the original number of motoneurons 
perish. Curare, as well as other neuromuscular junction blocking agents, have been shown 
to suspend NOCD in motoneurons presumably by increasing the number of synapses and 
thereby increasing the availability of target-derived NTFs at the neuromuscular junction 
(Oppenheim, 1991; Pittman and Oppenheim, 1978). In these experiments, approximately 
50% more motoneurons survive in curare-treated embryos than in control embryos. 

The chick has several advantages that make it a good choice as a model for FAS. 
Ethanol can be administered in exact doses to the developing embryo, and only the 
embryo's liver can remove the ethanol from the bloodstream. However, alcohol 
dehydrogenase does not begin in the developing chick until around E8 (Wilson et al., 
1984). Maternal influences are removed when using the chick embryo. Ethanol is cleared 
from the bloodstream by the mother in a mammalian system whereas the chick embryo is 
isolated as it develops. This model allows the investigator to observe direct effects of 
ethanol without interactions of maternal metabolism interfering. While chick development 
is different from mammalian gestation, this model allows researchers to study in vivo 
interactions in a developing organism that are not possible in a mammalian model. Another 
advantage for the research described in this document is that the chick embryo model has 
been used to study the ability of brain-derived neurotrophic factor (BDNf^ and glial cell 
line-derived neurotrophic factor (GDNF) to regulate NOCD in motoneurons of the chick 
embryo spinal cord (Oppenheim et al., 1995; Oppenheim et al., 1992). It is important to 



note that these experiments would be impossible to perform in mammals since exogenous 
NTFs can not be administered individually to developing fetuses. Oppenheim's laboratory 
has performed experiments using a variety of NTFs to study their effects on NOCD. In 
these experiments, NTFs are applied directly to the membranes of the developing embryo 
through windows in the outer egg shell. Replicating these experiments in mammals would 
require extensive surgical procedures that would undoubtedly have an adverse effect on 
fetal and maternal survival. Some of the present experiments utilize the chick embryo for 
all of the advantages described above. 

Motor System and Ethanol 
There is direct evidence that warrants further investigation into the possible effects 
that ethanol may exert on motoneurons. In both the human and in animal models, previous 
studies have determined that ethanol damages developing muscle (Adickes and Shuman, 
1983; Nyquist-Battie et al, 1987). In the human, these cases described flaccid, hypotonic 
neonates which exhibited major muscle structural deficiencies including hypotrophy, 
dominance of type II fibers, and sarcomeric dysplasia (Adickes and Shuman, 1983). 
Prenatal exposure to ethanol in the guinea pig resulted in structural malformations of the 
gastrocnemius muscle including vacuolated sarcoplasmic reticula, enlarged lipid droplets, 
decreased glycogen, and mitochondrial abnormalities (Nyquist-Battie et al., 1987). Proper 
muscle fiber maturation is dependent on concurrent development and innervation by 
motoneurons. Therefore, the possibility exists that deficiencies noted above are due to an 
underlying effect that ethanol exerts on developing motoneurons (Ishiura et al., 1981). It is 
equally likely that ethanol may exert some direct effect upon developing muscle. 
Accordingly, a limited analysis of muscle neurotrophic activity in chick leg muscle is 
undertaken in the present studies. 

Motor System and NTFs 
The developing motor system is dependent on many different NTFs for proper 
growth. Two of the NTFs that developing motoneurons encounter are BDNF and GDNF. 



BDNF is a member of the neurotrophin family of NTFs which includes nerve growth 
factor (NGF), neurotrophin-3 (NT -3), and NT-4/5. BDNF is a 1 18 amino acid residue 
polypeptide (Hag et al., 1994) that forms homodimers to attain its active form and binds 
with high affinity to tyrosme receptor kinase B (trkB; Klein et al., 1991). Previous 
research has identified two major pathways that arc initiated by autophosphorylation of trk 
(Stephens et al., 1994; Tolkovsky, 1997). One of these pathways leads to activation of the 
MAP kinase cascade and may initiate neurite outgrowth, transcription, or cellular 
hypertrophy (Stephens et al., 1994). The other pathway leads to the activation of akt (a 
serine/threonine kinase) and may initiate neurite outgrowth, survival, and receptor 
internalization (Tolkovsky, 1997). Developing skeletal muscle produces BDNF, which is 
known to support motoneuron survival during development by suspending NOCD in a 
subset of the developing motoneuron pool and to protect motoneurons of both the chick 
and rat from degenerating after lesion (Oppenheim et al., 1992; Sendtner et al., 1992; Yan 
et al., 1992). It is important to not that the addition of recombinant factors such as BDNF 
does not rescue all motoneurons in the developing motor column. BDNF is also expressed 
by the hippocampus, adrenal gland, and whole brain during rat development (Maisonpierre 
etal., 1990). 

The other NTF used in the present studies of chick development, GDNF, is a 
member of the transforming growth factor 6 superfamily and naturally occurs as a 
homodimer with a molecular weight of 40-45 kD (each molecule 134 amino acid residues; 
(Lin et al., 1993). GDNF and its receptors, GDNFRcc and c-ret, form a complex that 
allows c-ret to transduce intracellular signals from GDNF (Jing et al., 1996; Treanor et al., 
1996). Prior to binding with c-ret, GDNFR^ acts as a ligand-binding protein by binding 
GDNF (Jing et al., 1996). The GDNFRoc/GDNF complex then forms a complex with c- 
ret— the only molecule ol' the complex capable of producing intracellular signals (Jing et al., 
1996; Rosenthal, 1997; Treanor et al., 1996). The signal transduction pathways initiated 
by c-ret activation include the MAP kinase pathway (Worby et al., 1996) and the 



7 

Ras/ERK2 pathway (van Weering and Bos, 1997). MAP kinase and ERKs are proteins 
known to be involved in gene expression (Hazzalin et al., 1997; Mucsi et al., 1996). 
During rat development, GDNF mRNA is expressed by mesenchymal cells and in 
developing skeletal muscle begmning at E15, and in developing skin beginning at E17 
(Nosrat et al., 1996; Trupp et al., 1995; Wright and Snider, 1996). Peripherally in the rat, 
GDNF is expressed in the teeth, tongue, retina, nasal cavity, ear, kidney, and 
gastrointestinal tract during vaiious stages of development (Nosrat et al., 1996). Centrally, 
GDNF is expressed in the striatum, hippocampus, and cerebellum beginning at E15, and in 
the trigeminal motor nucleus (E17) and cortex (postnatal day 7). Generally, populations 
that are responsive to GDNF express c-ret. These populations include substantia nigra 
dopaminergic neurons (Trupp et al., 1995), spinal motoneurons (Pachnis et al., 1993; 
Tsuzuki et al., 1995), and certain subpopulations of the peripheral ganglia (Pachnis et al., 
1993; Tsuzuki et al., 1995). A small segment of Purkinje neurons does exhibit sensitivity 
to GDNF early in development before expression of c-ret commences (Nosrat et al., 1997), 
thus implying that GDNF might have the ability to signal through a receptor other than c- 
ret. During chick embryogenesis, c-ret mRNA is expressed in the Wolffian duct and 
ureteric bud, the enteric, dorsal root, sympathetic and facioacoustic ganglia, and the ventral 
spinal cord (Schuchardt et al., 1995). 

Neuroprotection 
NTFs have been shown to protect against insults such as hypoxia, hypoglycemia, 
and changes in calcium homeostasis. Examples of neuroprotection by polypeptide growth 
factors include epidemial growth factor protection of whole brain neuronal cultures from 
anoxia (Pauwels et al, 1989), NGF protection of rat hippocampal and human cortical 
neurons from hypoglycemia (Cheng and Mattson, 1991), and bFGF prevention of thalamic 
degeneration following cortical infarction (Yamada et al., 1991). GDNF is particularly 
potent in protecting neurons from a variety of conditions that normally result in death. 
Such insults and environmentally-produced conditions include NOCD (Oppenheim etal.. 



8 

1995), 6-OHDA lesion (Choi-Lundberg et al., 1997; Kearns and Gash, 1995; Tomac et 
al., 1995), and axotomy (Gimenez y Ribotta et al., 1997; Houenou et al, 1996; 
Oppenheim et al., 1995). While NOCD is not an insult in the sense that 6-OHDA lesion is, 
it does result in neuronal death and NTFs such as GDNF do prevent it from proceeding in a 
subset of the developing motor pool in live animals. Like GDNF, BDNF is effective in 
providing neuroprotection from events that normally result in neuronal death. However, 
different neuronal populations are protected by BDNF. For example, BDNF protects 
against ischemia-induced cell death in rat hippocampal slice cultures (Pringle et al., 1996), 
and prevents NOCD in some motoneurons (Oppenheim et al., 1992) and apoptotic death in 
PC12 cells (Jian et al., 1996) and cultured rat cerebellar granule neurons (Kubo et al., 
1995). The fact that both GDNF and BDNF provide such potent support for developing 
and injured neurons suggests that both could protect motoneurons against toxic events 
produced by ethanol. 

Neuroprotection from ethanol has been studied previously, but mostly in culture. 
Examples of this phenomenon include NGF protection of cultured dorsal root ganglion 
(DRG) neurons (Heaton et al., 1993) and septal neurons (Heaton et al., 1994) and basic 
fibroblast growth factor (bFGF) protection of cultured septal and hippocampal neurons 
(Heaton et al., 1994). Additionally, both NGF and bFGF protect cultured cerebellar 
granule cells from ethanol -induced cell death (Luo et al., 1997). Neuroprotection afforded 
by NGF and bFGF was found to require both protein and RNA synthesis which suggests 
that neuroprotection is related to a signal that the NTF receptor sends to the nucleus of the 
cell (Luo et al., 1997). GDNF has been shown to protect rat organotypic cultures of 
cerebellar Purkinje cells from ethanol neurotoxicity (McAlhany et al., 1997). To date, the 
only in vivo demonstration of NTF neuroprotection from ethanol toxicity is protection of 
choline acetyltransferase activity by NGF (Brodie et al., 1991). The reason that NTF 
neuroprotection is important for the study of FAS is that many of the toxic events that are 
prevented by NTFs in culture arc implicated as potential mechanisms for ethanol toxicity in 



9 

the nervous system. Mechanisms for ethanol toxicity that are suggested by previous 
research include hypoxia, hypoglycemia, and changes in calcium homeostasis (Alturaet 
al., 1983; Snyder et al., 1992; Webb et al., 1995). If these insults are indeed responsible 
for ethanol's toxic effects, NTFs might protect the developing nervous system from 
damage. 

Rat Model of FAS 

The rat is the most widely used model in FAS research. However, a caveat of 
using the rat as a model is the relative gestational period. Rat prenatal development is 
approximately equivalent to the first two trimesters of human development (Goodlett et al., 
1993). A major event of prenatal development in humans is the brain growth spurt (BGS), 
when many functional synapses are made in the nervous system. In rats, this event occurs 
postnatally from P4-P10 (West, 1987). Therefore, experiments that wish to mimic third 
trimester ethanol exposure in humans must incorporate the BGS. This objective requires 
postnatal ethanol exposure in rats. Exposure to ethanol during the BGS produces deficits 
that demonstrate the sensitivity of the CNS to ethanol during this period. Postnatal 
exposure in the rat produces deficits that are different from those seen following prenatal 
exposure. For example postnatal ethanol exposure can produce loss of cerebellar Purkinje 
cells (Bonthius and West, 1990; Bonthius and West, 1991; Goodlett and West, 1992; 
West, 1986; West et al., 1990). Since postnatal ethanol exposure damages neuronal 
populations known to be damaged in FAS, it does serve as a model for FAS. 

A caution of any postnatal exposure paradigm is that maternal metabolism of 
ethanol is removed and the subjects are exposed to ethanol in the same manner as adults 
(i.e., only the neonatal liver removes ethanol from the bloodstream). Alcohol 
dehydrogenase (ADH) activity begins in the rat on approximately gestational day 15 
(Boleda et al., 1992; Tietjen et al., 1994). Fetal ADH has very low activity in comparison 
to adult ADH, which suggests that metabolism of ethanol during pregnancy is completed 
almost entirely by maternal ADH. Between P20 and P39 all subclasses of ADH reach 



10 

100% of adult activity (Boleda et al. , 1992) . Another difficulty is that suckling rats will not 
readily consume ethanol because their entire diet consists of mother's milk. 

Two typical methods for ethanol deUvery to newborn rats are artificial rearing (AR) 
and vapor inhalation, both of which have advantages and disadvantages. AR is a surgical 
procedure which consists of fitting a neonatal pup with a gastric fistula and tube, 
maintaining the pups in cups placed in a heated water bath, and feeding the pup an artificial 
milk solution via the tube and fistula. The advantage of the AR method is that it provides 
constant nutrition and produces no damage to the mucous membranes of the subject. Major 
disadvantages of AR are that interaction between mother and pup is removed and that it can 
be stressful for the neonatal rat. The stress mduccd by AR produces gliosis in rat cortex 
(Ryabinin et al., 1995), although gliosis following postnatal exposure via intragastric 
intubation was observed (Goodlett et al, 1997). In this latter experiment, gliosis was not 
observed in control animals (Goodlett et al., 1997). The fact that the AR procedure per se 
may produce changes in brain structure indicates that results obtained using AR could be 
difficult to interpret. The ethanol vapor inhalation procedure consists of placing neonatal 
rats in a sealed chamber that contains circulating air and ethanol vapor. This procedure has 
been theorized to damage the mucous membranes of the lungs which could interfere with 
oxygen exchange and general metabolism (Ryabinin et al, 1995). However, no evidence 
supports this contention and lung damage has not been observed in rats exposed to ethanol 
vapors (Bauer-Moffet and Altman, 1975). Other methods for delivering ethanol to neonatal 
rats include delivery of ethanol through mother's milk by limiting the dam's liquid intake to 
ethanol-containing fluids, and exposing pups to ethanol vapor concurrently with the dam. 

Rat Septohippocampal System 
Many neuronal populations exhibit some susceptibility to the toxic effects of 
ethanol. As was mentioned above, a variety of neuronal types from brain regions such as 
the cerebellum (Cragg and Phillips, 1985), the septohippocampal system (Barnes and 
Walker, 1981; West and Pierce, 1986), the cerebral cortex (Miller, 1986), and the 



11 

oculomotor nucleus (Burrows et al., 1995) are known to be affected in some way by 
ethanol exposure. The hippocampus is an important structure with regard to memory and 
learning in humans and animals (Bunsey and Eichenbaum, 1996; Cohen and Squire, 
1980). Therefore, damage to the hippocampus observed in the rat following ethanol 
exposure may correspond to similar damage to the hippocampus in humans. Since learning 
and memory deficits are a common characteristic of FAS (Abel, 1995; Jones and Smith, 
1973; Streissguth et al., 1991), it is not surprising to find that the hippocampus is sensitive 
to ethanol and exhibits reduced cell number following ethanol exposure (Barnes and 
Walker, 1981). 

Neurotrophic Factors 
The neurotrophin family of NTFs plays a valuable role in the development of the 
nervous system through regulation of neuronal differentiation and survival, and 
maintenance of basic cellular processes. The neurotrophin family, as noted above, includes 
NGF (Levi-Montalcini, 1951), BDNF (Leibrocket al., 1989), NT-3 (Maisonpierreetal, 
1990), NT-4/5 (Berkemeier et al., 1991; Ip et al., 1992), and neurotrophin-6 (Gotz et al., 
1994). The trk family of receptors has been shown to be the high-affinity receptors for the 
neurotrophins (Martin-Zanca eL al., 1990). Trks that bind neurotrophins include trkA 
(Kaplan et al., 1991; Kaplan et al., 1991), trkB (Klein et al., 1990), and trkC (Cordon- 
Cardo et al, 1991). TrkA is the preferred receptor for NGF, but will bind both BDNF and 
NT-3. TrkB is the preferred receptor for BDNF and NT-4/5, but will bind NT-3. TrkC is 
the preferred receptor for NT-3. The neurotrophins regulate a number of peptides in the rat 
septohippocampal system, including other neurotrophins (Croll et al., 1994). For 
example, NGF, BDNF, and NT-3 induce cholme acetyl transferase activity (Alderson et al., 
1990; Auberger et al., 1987; Gnahn et al., 1983; Nonner et al., 1996); BDNF increases 
NT-3 activity (Lindholm el al., 1994) ; and BDNF and NT-3 enhance synaptic transmission 
in Shaffer collateral-CA 1 hippocampal synapses (Kang and Schuman, 1995). All of these 
results indicate the importance of the neurotrophins in this brain region. 



12 

Gene Deletion Studies 
Studies of gene-deleted "knockout" mice also suggest the importance of 
neurotrophins and other NTFs m proper nervous system development. Knockout studies 
consist of deleting a gene from the mouse genome by homologous recombination (Smithies 
et al., 1985). Following this procedure, the mice are allowed to develop and are 
subsequently compared to control mice. All neurotrophin and neurotrophin receptor 
knockout animals die relatively early in development except for NT -4/5 animals (Conover 
and Yancopoulos, 1997). Specifically, NGF knockout mice exhibit reduced numbers of 
superior cervical ganglion, trigeminal ganglion, and DRG neurons (Crowley et al., 1994). 
BDNF knockout mice have decreased numbers of trigeminal ganglion, geniculate ganglion, 
nodose-petrosal ganglion, vestibular ganglion, spiral ganglion, and DRG neurons 
(Conover et al., 1995; Ernfors et al., 1994). NT-3 knockout mice display fewer superior 
cervical ganglion, trigeminal ganglion, nodose-petrosal ganglion, vestibular ganglion, 
spiral ganglion, DRG neurons, and spinal cord motoneurons (Ernfors et al., 1995; Kucera 
et al., 1995). NT -4 knockout mice are similar to BDNF knockouts and have reduced 
geniculate ganglion, nodose-petrosal ganglion, vestibular ganglion, and DRG neurons 
(Conover etal., 1995). 

Knockouts of receptors of neurotrophins also support the idea that they are 
important in proper ner\'Ous system development. TrkA knockout mice show the same 
pattern of neuronal loss that the NGF knockout mice have (Smeyne et al., 1994). TrkB 
knockouts have reduced numbers of spinal cord and facial motoneurons, and exhibit 
reduced numbers of trigeminal ganglion, nodose-petrosal ganglion, and DRG neurons 
(Klein et al., 1993). This result is especially significant in light of the fact that BDNF 
knockout mice did not exhibit motoneuron deficits. This discrepancy between the two 
knockout studies suggests that trkB is important for motoneuron development and some 
other molecule, perhaps NT-3, can bind trkB to promote survival in the absence of BDNF. 
TrkC knockout mice exhibit reduced numbers of DRG neurons, and completely lack la 



13 

muscle afferents (Klein et al, 1994). All single gene neurotrophin knockout studies 
observed reduced DRG number. These results support earlier studies that found DRGs 
respond to many different NTFs and express the receptors for multiple neurotrophins 
(Buchman and Davies, 1993). 

Knockout mice have also been used to study GDNF and c-ret. GDNF-deficient 
animals exhibit deficits in DRG, sympathetic and nodose neurons, but not in hindbrain 
noradrenergic or midbrain dopaminergic neurons and completely lack the enteric nervous 
system, ureters, and kidneys. These animals did display a small yet significant (~20%) 
loss of spinal cord motoneurons (Moore et al., 1996). C-ret knockout mice do not contain 
reduced numbers of motoneurons, but do lack enteric nervous system and contain reduced 
numbers of parasympathetic neurons (Marcos and Pachnis, 1996). These studies 
demonstrate that NTFs other than the neurotrophins are important to the normal 
development of the CNS. 

While gene-targeting studies have provided invaluable information regarding the 
action of NTFs and their receptors in the nen'ous system, they must be interpreted with 
certain difficulties in mind. Knockout mice are not merely normal animals with one gene 
conveniently deleted. These organisms possess a number of developmental, physiological, 
and even behavioral processes that have been altered to compensate for the missing gene 
(Gerlai, 1997). For example, when one gene is eliminated from an organism, the 
transcription of other gene products may be altered. This change in transcription could 
conceivably ameliorate or exacerbate the effects of losing the gene. The many redundancies 
present in the genome may mask the effects of losing a single gene. Therefore, it would be 
difficult to determine whether the observed changes in the organism were due to loss of the 
gene of interest or to the change in genetic background. 

The knockout studies relate to the present experiments in that they provide a clue of 
what would happen lo the nervous system should a specific NTF or NTF receptor be 
removed. Recall that the neurotrophic hypoihesis— which is a driving force for this 



14 

research- suggests that removal or reduction of neurotrophic support would have a 
deleterious effect upon the developing nervous system. The above evidence from the 
knockout studies supports this idea. Since some populations are reduced in number in 
knockout mice, this suggests that a lack of proper neurotrophic support can reduce neuron 
number. Should ethanol effectively remove or reduce neurotrophic support from a given 
nervous system population, that population will undoubtedly be adversely affected. To 
determine whether this change in some aspect of neurotrophic support is indeed occurring, 
it is logical to examine populations that are known to exhibit cell loss due to ethanol 
exposure (e.g. the hippocampus). Knockout studies have not recorded any cell loss in the 
septohippocampal region. The fact that the septohippocampal system is a rich source for a 
variety of NTFs may provide an explanation for this apparent paradox. If one NTF is 
eliminated, other NTFs in the background may be able to provide sufficient support for the 
neurons to continue to survive. The basic idea that NTF support is critical for survival is 
important for the present studies. 

NTF Ontogeny in the Hippocampus 
The normal ontogeny of NGF, BDNF, and NT -3 in the developing rat 
hippocampus differs. NGF is expressed in rather low levels throughout embryonic 
development, increases somewhat at birth, and finally achieves its highest levels in the 
adult. BDNF is virtually undetectable throughout embryonic development, then increases 
at birth and continues to increase to its highest levels in the adult rat brain. NT -3 has high 
expression throughout development and decreases in the adult (Maisonpierre et al., 1990). 
The different temporal expression between individual neurotrophins might help developing 
neurons achieve correct synapses. The following hypothetical example illustrates this 
point: The high eaily expression of NT-3 might promote survival of neurons through a 
proliferative phase. Then, NGF expression could increase to induce differentiation. 
Finally, BDNF expression would signal the end of development and thus induce these 
hypothetical neurons to form synapses. //; vivo, neurotrophins have been shown to follow 



15 

distinct patterns throughout development. Buchman and Davies found that neurotrophins 
act in sequence during development to promote survival of DRGs (1993). Therefore, if the 
temporal sequence of neurotrophin expression were to change as a result of ethanol 
exposure, the normal innervation patterns in the hippocampus— as well as other parts of the 
developing CNS-could be altered. Such a change could have disastrous effects on the 
hippocampus and its ability to properly encode new memories. 

The ontogeny of tri: receptors in the developing brain follows the ontogeny of the 
neurotrophins. In the septum~a brain structure of which the hippocampus is a target- 
higher levels of trkA niRNA were detected at 2 and 4 weeks than at 1 weeks of age 
(Ringstedt et al, 1993). TrkA is not expressed in the hippocampus under normal 
circumstances //; vivo (Martin-Zanca et al, 1990). TrkB is expressed widely in the CNS 
and is first detectable in the mouse at E8.5 (Klein et al., 1990). TrkB is expressed by the 
developing hippocampus and expression continues into adulthood (Klein et al., 1990). 
TrkC mRNA is detectable as early as E7.5 in the nervous system and is expressed at all 
stages of hippocampal development (Tessarollo et al., 1993). The above information 
demonstrates that developing hippocampal neurons are responsive to BDNF and NT -3 and 
that these proteins, plus their receptors, are expressed during prenatal and early postnatal 
rat development. Thus, all of these proteins were active during the periods of ethanol 
exposure employed in the present study. 

Neurotrophins and Ethanol 
Fundamental responses to neurotrophins and production of NTFs are altered 
following prenatal ethanol exposure in rat pups. The neurotrophins are not the only NTFs 
produced by the hippocampus. Other factors, such as bFGF, are synthesized there and 
could affect these neurons (Emfors et al., 1990). Cultures of hippocampal neurons derived 
from rats prenatal! y exposed to ethanol do not respond to NTFs as well as neurons in 
control cultures (Heaton et al., 1995b). This result suggests that NTF receptor expression 
may be decreased in response to prenatal ethanol exposure. However, another logical 



16 

explanation for this result is that the expression of less active form of NTF receptor has 
increased relative to a normal fomi. Truncated trk receptors are similar to normal trk 
receptors except they lack the catalytic domain that starts the intracellular signal transduction 
cascade following neurotrophin binding. These receptors are normally expressed in greater 
abundance than their active counterpaits in adult animals. During development their 
expression increases relative to the active trk receptor until reaching the level of expression 
found in the adult. Other studies have found altered neurotrophic activity as a result of 
ethanol exposure. For example, chronic prenatal ethanol treatment (CPET) in the rat 
increases neurotrophic activity (a gross measure which includes both neurotrophin and 
other NTF activity) in extracts made from the hippocampus on P21 and cultured on DRG 
neurons (Heaton et al., 1995c). The increase in neurotrophic activity suggests an increase 
in NTF expression as a result of prenatal ethanol exposure, but no single NTF is implicated 
by this study since DRGs respond to a variety of NTFs. Postnatal ethanol exposure 
reduces neurotrophic activity of P21 hippocampal extracts (Moore et al., 1996), a result 
opposite to that of prenatal exposure (Heaton et al., 1995c). All of these results suggest 
that both NTF and receptor might play an essential role in ethanol toxicity. 

Hypotheses 
As was mentioned previously, all experiments described in this document were 
designed to understand some aspect of how ethanol and NTFs are interrelated in producing 
the deficits observed in models of FAS. The experiments of this project were performed to 
test the following hypotheses: (1) (a) We hypothesize that ethanol will reduce motoneuron 
number in the absence NOCD; (b) We hypothesize that ethanol will reduce motoneuron 
number at period of development that follo\vs the period for NOCD; (2) We hypothesize 
that exogenous NTFs will provide in vivo protection for motoneurons exposed to ethanol; 
and (3) We hypothesize that CPET and early postnatal ethanol treatment (EPET) will alter 
the gene expression of neurotrophins and/or their receptors in the hippocampus of treated 
rat pups. Analysis of hypothesis la was undertaken to further describe the motoneuron 



17 

loss observed following ethanol exposure from E4 to El 1 in chick embryos. Specifically, 
we wanted to determine whether ethanol acted to increase NOCD or provide direct 
neurotoxicity. Ethanol was shown to reduce motoneuron number during this time period 
which encompasses the period for NOCD (Heaton and Bradley, 1995). Additionally, 
neurotrophic content of the developmg muscle was analyzed to compare ethanol exposure 
from ElO to E15 to an earlier study in this laboratory which found that ethanol exposure 
from E4 to E8 reduced neurotrophic content of developing limb tissue (Heaton and 
Bradley, 1995). Analyzing neurotrophic content of muscle from embryos exposed to 
ethanol from ElO to E15 allowed us to relate any deficit in motoneuron number observed in 
that time period, to any possible change in neurotrophic support. The number of apoptotic 
cells present during ethanol exposure from El to E15 was analyzed to find whether 
ethanol exposure from ElO to E15 mduced apoptosis among motoneurons. Analysis of 
hypothesis 2 was executed to detennme whether NTFs could modulate ethanol toxicity in 
vivo. Analysis of hypothesis 3 was perforaied to ascertain whether ethanol could modulate 
the genetic expression of NTFs in a living organism. The chapters that follow describe the 
experiments perfomied to achieve these hypotheses and provide the results of these 
analyses. Further, the results are discussed critically with implications for future research 
and mechanisms for ethanol toxicity suggested. 



CHAPTER 2 

CHARACTERIZATION OFMOTONEURON SURVIVAL AND CELL DEATH 

FOLLOWING ETIiANOL EXPOSURE AND CURARE ADMINISTRATION, AND 

AFTER THE PERIOD FOR NATURALLY OCCURRING CELL DEATH 

Summary 
The study described below was conducted as a continuation of a previous study in 
which we found reduced motoneuron number in lumbar spinal cord of the chick embryo 
following chronic ethanol administration from embryonic day 4 (E4) to El 1. We sought to 
determine whether this reduction was due to primary ethanol toxicity or to enhancement of 
naturally occurring cell death (NOCD) and to determine whether administration of ethanol 
at a later period of development could also reduce motoneuron number. Eariier studies 
have shown that curare suspends NOCD in the chick embryo (Pittman and Oppenheim, 
1978). By administering both ethanol and curare to these embryos from E4 to El 1 and 
examining the lumbar spinal cord on E12, we determined that ethanol was directly toxic to 
motoneurons and reduced motoneuron number in the absence of NOCD. By administering 
ethanol from ElO to E15 and examining the lumbar spinal cord on E16, we determined that 
ethanol can reduce motoneuron number without altering the overall morphology of the 
spinal cord during more than one stage of chick embryo development. We also determined 
that ethanol toxicity is not dependent on NOCD. In additional experiments, we 
demonstrated that ethanol does not affect the neurotrophic content of chick muscle and does 
not appear to induce apoptosis in developing motoneurons when it is administered from 
ElO to E15. 

Introduction 
Over the last two and one-half decades, much evidence has been gathered regarding 
the effects ethanol exerts in the developing nervous system (Barnes and Walker, 1981 ; 



18 



19 

Jones and Smith, 1973; Miller, 1986; Streissguth et al., 1991; West, 1986). Fetal alcohol 
syndrome (FAS) is diagnosed in 1-2 out of every 1000 live births in the United States and 
is characterized by low birth weight, decreased memory and learning, hyperactivity, facial 
dysmorphia, and lowered IQ (Abel, 1995; Jones and Smith, 1973; Streissguth et al., 
1991). Among heavy drinkers, the incidence of FAS is much greater with a 4.3% 
diagnosis rate (Abel, 1995). Evidence suggests that these deficits are permanent and do not 
lessen as the patient ages (Streissguth, 1993). These observations led to the assertion that 
maternal consumption of ethanol is the leading known cause of mental retardation in the 
Western Hemisphere (Bonthius and West, 1988). 

Many neuronal populations exhibit some susceptibility to the toxic effects of 
ethanol. A variety of neuronal types from brain areas such as the cerebellum (Cragg and 
Phillips, 1985), the septohippocampal system (Barnes and Walker, 1981) (West and 
Pierce, 1986), the cerebral cortex (Miller, 1986), and the oculomotor nucleus (Burrows et 
al., 1995) are known to be affected by ethanol exposure. Still other populations that have 
not been so intensely investigated demonstrate vulnerability to ethanol. These regions 
include the substantia nigra (Shetty et al., 1993), chief sensory trigeminal nucleus (Miller 
and MuUer, 1989), red nucleus (Zajac et al., 1989), and motoneurons of the spinal cord 
(Heaton and Bradley, 1995). Of particular interest to the present studies is the fact that 
motoneurons are affected by ethanol. A previous study from this laboratory found that 
motoneuron number was reduced by ethanol administration from E>^ to El 1 (Heaton and 
Bradley, 1995). At that time, we hypothesized that ethanol might be exacerbating NOCD. 
The period for NOCD for motoneurons of the entire spinal cord extends from 
approximately E6 to E9 (Pittman and Oppenheim, 1978). Motoneuron number is known to 
peak around E5.5 to E6 (Pittman amd Oppenheim, 1978) and proliferation of motoneurons 
continues until E6 (HoUyday and Hamburger, 1977). During the period of NOCD, nearly 
half of the original number of motoneurons perish. Curare, as well as other neuromuscular 
junction blocking agents, have been shown to suspend NOCD in motoneurons (Pittman 



20 

and Oppenheim, 1978). In these experiments, approximately 50% more motoneurons 
survive in curare-treated embryos than in control embryos. 

In addition to the above, there is more direct evidence that warrants further 
investigation into the possible effects that ethanol may exert on motoneurons. Previous 
research in other laboratories has determined that ethanol damages developing muscle in 
both the human and in animal models (Adickes and Shuman, 1983; Nyquist-Battie et al., 
1987). In the human, these cases described flaccid, hypotonic neonates which exhibited 
major muscle structural deficiencies including hypotrophy, dominance of type II fibers, and 
sarcomeric dysplasia (Adickes and Shuman, 1983). Prenatal exposure to ethanol in the 
guinea pig resulted in structural malformations of the gastrocnemius muscle including 
vacuolated sarcoplasmic reticula, enlarged lipid droplets, decreased glycogen, and 
mitochondrial abnormalities (Nyquist-Battie et al., 1987). Since proper muscle fiber 
maturation is dependent on concurrent development and innervation by motoneurons 
(Ishiura et al., 1981), the possibility exists that the deficiencies noted in the above cases are 
due to an underlying effect that ethanol exerts on developing motoneurons. 

Previous studies have shown that ethanol affects chick embryo development in a 
manner similar to both the human and the rat. Chicks have been shown to exhibit reduced 
brain size, weight, DNA and protein synthesis (Pennington and Kalmus, 1987), and 
reduced neurotransmitter synthesis (Brodie and Vernadakis, 1990; Swanson et al., 1994) 
following developmental ethanol exposure. One advantage of using a chick model to study 
ethanol is its simplicity. Ethanol can be administered in exact doses to the developing 
embryo. Another advantage is that maternal influences are removed when using the chick 
embryo. Ethanol is cleared from the bloodstream by both mother and fetus in a mammalian 
system whereas the chick embryo is isolated as it develops. The chick model allows the 
investigator to observe direct effects of ethanol without possible interactions of maternal 
metabolism interfering. The embryos seem to tolerate slight invasions into their 
environment quite well as long as the underlying membranes are not disrupted. While 



21 

chick development is clearly different from mammalian gestation, this model allows 
researchers to study in vivo interactions in a developing organism that are just not possible 
in a mammalian FAS model. 

The present experiments were performed to determine whether ethanol exerted a 
toxic effect when NOCD is suspended (Curare-Ethanol Coadministration) and whether 
administering ethanol dunng a later period of development (Late Exposure)— after the 
period of cell death- would differ from administration earlier in development. Suspension 
of NOCD did not hinder ethanol' s ability to reduce motoneuron number. This result 
suggests that ethanol acts by a mechanism other than exacerbation of NOCD, either by 
direct toxicity, motoneuron loss due to a change in neurotrophic support, or a combination 
of the two. The Late Exposure study found that ethanol administration from ElO to E15 
reduced motoneuron number and that exposure to ethanol did not reduce the neurotrophic 
activity of chick limb muscle in comparison to Saline treated embryos. This latter analysis 
was undertaken to compare the results of ethanol exposure from ElO to E15 to an earlier 
study in this laboratory which found that ethanol exposure from E4 to E8 reduced 
neurotrophic content of developing limb tissue (Heaton and Bradley, 1995) and to 
determine if altered neurotrophic support is responsible for the observed motoneuron 
reduction. Loss of neurotrophic support would suggest a possible mechanism for 
motoneuron loss due to ethanol exposure at this period of development: reduction of 
target-derived NTFs. Additionally, an analysis of apoptotic motoneurons in the lumbar 
spinal cord failed to find evidence that ethanol exposure from ElO to E15 induced apoptosis 
among motoneurons. 

Materials and Methods 
Subjects 

White Leghorn chick eggs were obtained from the University of Horida Poultry 
Science Department. Eggs were placed in a Marsh incubator and maintained at 37°C and 



22 

70% relative humidity until B4. At that time, the eggs were moved to a forced draft turning 
incubator, maintained at the same conditions indicated above, and divided into groups. 
Curare-ethanol coadministration 

For the Curare-Ethanol Coadministration study, 5 groups were utilized: 
Uninjected, Ethanol, Salme, Curare, and Curare+Ethanol. The data from all Ethanol 
embryos and 4 of the 10 Saline embryos were obtained from an earlier study (Heaton and 
Bradley, 1995). Ethanol and saline injection began on E4 and continued daily through 
Ell. Curare injection began on E6 and continued daily through Ell. Ethanol and curare 
injections were initiated on different days in order to replicate previous research and to 
coincide curare administration with the onset on NOCD in the lumbar spinal cord (E6) 
(Pittman and Oppenheim, 1978). Since cell death in the lumbar section of the spinal cord 
does not begin until E6 (HoUyday and Hamburger, 1977; Pittman and Oppenheim, 1978), 
curare administration before this time point would be without effect. In addition, the 
present experiment found the combination of curare and ethanol to be quite toxic to the 
developing embryos; therefore, further loss of embryos was minimized by starting curare 
administration on E6. All embryos were sacrificed by decapitation on E12, the lumbar 
section of the spinal cord removed, and prepared for histology. 
Late exposure study 

A separate study was conducted to assess the effects of ethanol during a later 
exposure period when motoneuron number is relatively stable. The Late Exposure study 
had 2 groups: Ethanol and Saline. Embryos received daily injections of either ethanol or 
saline from ElO to E15. At E16, embryos were removed from the eggs, sacrificed by 
decapitation, and the lumbar section of the spinal cord removed and prepared for histology. 
An Uninjected control group was not used in the Late Exposure study because in previous 
studies, our laboratory has shown that saline injection does not adversely affect embryonic 
development and has no effect on motoneuron number in the developing chick spinal cord 
(Heaton and Bradley, 1995). 



23 



Injections 

Curare-ethanol coadministration study 

As stated above, five experimental groups were used in this study: Ethanol, Saline, 
Uninjected, Curare, and Curare+Ethanol. Ethanol and saline injections were administered 
daily from E4 through Ell. Ethanol embryos received 150 ^wl of 20% w/v ethanol (30 mg 
ethanol per day), dissolved in a 0.9% w/v nonpyrogenic saline vehicle, through a pinhole 
in the shell into the airspace. Previous work in our laboratory has determined that this 
concentration of ethanol produces blood ethanol counts that peak at 225 mg/dl by El 1 
(Heaton and Bradley, 1995). Saline embryos received 150 jaI of the 0.9% w/v 
nonpyrogenic saline vehicle. Uninjected embryos received no injections, but were handled 
daily in a manner similar to the other groups. Curare injection, which occurred from E6 
through Ell, involved creating a pinhole directly over the embryo in addition to the pinhole 
created in the airspace. The airspace was then allowed to shift to a position above the 
embryo and 150/^1 of 16.67 mg/ml tubocurarine chloride (Sigma) was injected into that 
space above the embryo (2.5 mg curare per day). Curare+Ethanol embryos were given 
ethanol injections from E4 to El 1 and curare injections from E6 to El 1 as described above. 
The embryos were allowed to sit in a Marsh incubator for a period of one hour following 
ethanol injection on days when two injections were delivered to the same embryo. This 
delay in injection time was necessary to ensure that the ethanol was absorbed through the 
inner shell membrane within the airspace before the eggs were turned on their side for the 
curare administration. Also, the two injections administered in this study represent a 
significant volume (300 piL) for the embryonic system to incorporate on a daily basis. The 
delay between injections, therefore, allowed absorption of the volume of ethanol before an 
equal volume of curare solution was presented to the embryo. Pinholes created by the 
injection process were sealed with paraffin immediately following injection to prevent 
evaporation and/or leakage of the ethanol and curare. The eggs were then returned to the 
turning incubator. 



24 

Late exposure study 

Ethanol and saline injections were administered daily from ElO through E15. 
Ethanol embryos received 150 /^l of 30% w/v ethanol (45 mg ethanol per day), dissolved in 
a 0.9% w/v nonpyrogenic saline vehicle, through a pinhole into the airspace. Since 
embryos at this later stage of development do have the ability to clear ethanol from the 
bloodstream (Wilson et al., 1984), a larger dose of ethanol was utilized in this study in 
order to achieve blood ethanol concentrations similar to those observed in embryos exposed 
to ethanol from E4 to El 1. Peak blood ethanol concentration in this portion of the study 
ranged from 250 to 300 mg/dl and trough levels were below 30 mg/dl. Saline embryos 
received 150 pi\ of the 0.9% w/v nonpyrogenic saline vehicle. Pinholes created by the 
injection process were sealed with paraffin following injection to prevent evaporation 
and/or leakage of the ethanol. The eggs were then returned to the turning incubator, as 
above. 

Dissections and Histological Procedures 
Curare-ethanol coadministration study 

Embryos of all experimental groups (Ethanol, Saline, Unmjected, Curare, and 
Curare+Ethanol) were sacrificed by decapitation on E12 and the lumbar section of the 
spinal cord removed. Following dissection, the E12 spinal cords were placed in Bouin's 
fixative for 24 hours and then embedded in paraffin. Spinal cords were then cut mto 12 
]Am coronal sections, mounted onto glass slides, and stained with hematoxylin and eosin. A 
total of 33 embryos were used in this study: Ethanol (n=6), Saline (n=10), Uninjected 
(n=6). Curare (n=6), and Curare+Ethanol (n=5). 
Late exposure study 

Embryos of both experimental groups (Ethanol and Saline) were sacrificed by 
decapitation on E16 and the lumbar section of the spinal cord removed. The vertebrae of 
the spinal cords were cut along the dorsal surface to expose the nervous tissue and allow 
the fixative to adequately penetrate the tissue. Following dissection, the E16 spinal cords 



25 

were placed in Bouin's Fixative for 14 to 21 days to allow the vertebrae to decalcify (Li et 
al, 1994). The tissue was then embedded in paraffin, cut into 12 pim coronal sections, 
mounted onto glass slides, and stained with hematoxylin and eosm. 7 Ethanol and 6 Salme 
embryos were used to complete this study. 

Motoneuron Counts 

Motoneuron counts were completed following methods described previously 
(Hamburger, 1975; Heaton and Bradley, 1995). Briefly, a uniform area encompassed by 6 
dorsal root ganglia (DRG) was noted on each embryo which ensured that a similar area was 
counted in each subject. Starting from the most rostral section included in the 6 DRG 
region, motoneurons in the lateral motor column of one side of every tenth section were 
marked onto paper using a camera lucida. At 400X magnification, motoneurons were 
identified in the lateral motor column by their large size, dark cytoplasm, and nucleolus. 
Laterality was maintamed throughout each individual embryo, but chosen at random before 
begmning the counting process. Previous studies have shown that there is no difference 
between the number of motoneurons contained in the right and left sides of the spinal cord 
(Pittman and Oppenheim, 1979). It should also be noted that each embryo was coded so 
that the experimenter had no knowledge of its experimental treatment until the study was 
completed. Motoneuron counts reported below are actual counts generated by the above 
procedure and are not corrected to estimate total motoneuron number of the lumbar spinal 
cord. 

Pre-Cell Death Ethanol Exposure 

In order to clanfy the results of the Coadministration Study, we administered 
ethanol and saline to chicks from E4 to E5 and assessed motoneuron number on E12. This 
analysis was performed to determine whether ethanol reduces motoneuron number during 
the time period where ethanol was administered, but curare administration had not yet 
commenced. Chick eggs were incubated as described previously and placed into two 
groups; Ethanol and Saline. On E4 and E5 Ethanol embryos received 150 pi\ of 20% w/v 



26 

ethanol solution (30 mg) and Saline embryos received 150 pi\ of the saline vehicle. No 
injections were administered from E6 to El 1 and on E12, the embryos were sacrificed by 
decapitation, the lumbar spinal cord removed, and prepared for histology. 

Motoneuron Size and Spinal Cord Length Analyses 

Motoneuron size and spinal cord length were measured to determine whether 
ethanol had altered any general characteristics of the motoneuronal system. Motoneuron 
size was determined by measuring the diameter of 10 random cells in the same rostral- 
caudal position of the region of each embryonic spinal cord. In E12 embryos 
(Coadministration Study) the section exactly ISOO/^m following the beginning of the 
lumbar spinal cord as determined by the 6 DRG region described above, was sampled. In 
E16 embryos (Late Exposure Study) the section exactly 2400 jAm following the beginning 
of the lumbar spinal cord, was sampled. Four embryos from each experimental condition 
were analyzed for a total of 40 cells per condition. Spinal cord length was determined by 
counting the number of sections present in each embryo following determination of the 
boundaries of the lumbar spinal cord by the anatomical methods described previously and 
multiplying this number by the section thickness (12 ><m). 

Crude Muscle Extract Study 
Extract preparation 

In order to determine whether neurotrophic content of chick leg muscle is affected 
by ethanol exposure during the late exposure period (ElO to E15), we analyzed the activity 
of crude muscle extracts on E6 spinal cord cultures. This analysis was undertaken to 
compare ethanol exposure from ElO to E16 to ethanol exposure from E4 to E8-where a 
reduction in neurotrophic activity of developing muscle tissue following ethanol exposure 
was observed. Embryos were treated with ethanol or saline and incubated as described in 
the Subjects section above. Briefly, Ethanol and Saline embryos received injections of 
30% w/v ethanol (45 mg per day) or the saline vehicle, respectively, daily from ElO to 
E15. On E16, embryos were removed from the egg, sacrificed by decapitation, and the 



27 

muscle dissected away from the thigh region of each leg. The muscle tissue was flash- 
frozen on dry ice and then stored at -70°C until extract was prepared. Extract was prepared 
by homogenizing the tissue for about 15 seconds in F-12 media (BRL) supplemented with 
0.7% fungizone, 1.0% penicillin-streptomycin, and 200 mM glutamine. After 
homogenization, the extract was centrifuged for 20 minutes at 35,000 rpm at 4°C. The 
supernatant was collected and assayed for protein content (Bradford, 1976). All samples 
were diluted to 200 pcglml protein, 10% fetal bovine serum (FBS) added, and then added to 
mixed spinal cord cultures which were prepared as described below. 
Spinal cord cultures 

Three experimental groups were analyzed in this portion of the study: Ethanol, 
Saline, and Negative Control (NC). Ethanol cultures consisted of E6 lumbar spinal cord 
cultures grown in the presence of muscle extract obtained from embryos exposed to ethanol 
from ElO to E15. Saline cultures were grown in the presence of muscle extract obtained 
from Saline embryos and NC were grown in the presence of regular culture medium. 
Regular culture medium consisted of F-12 media supplemented as described above, with 
10% FBS added. The lumbar region of the spinal cord was dissected out of E6 embryos 
and incubated in 10% v/v trypsin and 5% v/v deoxyribonuclease 1 in 0.9% nonpyrogenic 
saline for 20 minutes at 37°C. Cells were disassociated by repeated titration in regular 
culture medium. The cells were grown in individual wells of 12-well Corning plates coated 
with 0.5 mg/ml polyomithine. Two hours after the cells were plated initial counts were 
completed by counting three representative areas from each culture well. Each culture had a 
Bellco glass slip with an enumerated grid affixed to the bottom. This allowed the 
experimenter to note the initial areas counted and return to these areas on subsequent days. 
Neurons were identified by their large size, and rounded, phase-bright appearance. 
Following 24 and 48 hours in culture, cell counts were again obtained from each culture 
and in addition, the number of cells expressing a neurite (at least two cell diameters in 
length) were noted. 



28 

Assessment of Apoptotic Cells 

Following ethanol exposure to embryonic chicks as described previously, spinal 
cord tissue was examined and apoptotic cells were identified by methods described 
previously (Homma et al., 1994). Briefly, at 400X magnification cells fitting the following 
criteria were determined to be undergoing apoptosis. First, cells with chromatin and 
cytoplasmic condensation— the hallmark of apoptosis— were identified. Since these 
processes are rather fast, it was unlikely that such cells would be observed. Therefore, a 
second criterion— where apoptotic debris was identified— was used to make a positive 
identification of an apoptotic cell. Since this portion of the study utilized an ethanol 
exposure paradigm that began on ElO and ran through E15, tissue was examined at the 
following intermediate time points: E12 and E14. Both saline and ethanol-exposed 
embryos were examined so that a statistical comparison could be made between the two 
groups. It should be noted, however, that NOCD essentially ends on E9 (Pittman and 
Oppenheim, 1978). A uniform area encompassed by 6 DRG was noted on each embryo 
which ensured that a similar area was counted in each subject. Starting from the most 
rostral section included in the 6 DRG region, apoptotic motoneurons in the lateral motor 
column of one side of every fifteenth section were noted. This process led to an average of 
21 sections being examined per E12 embryo and 30 sections being examined in every E14 
embryo. After the entire animal was examined, candidate cells were reexamined to make 
sure that they truly fit the criteria described above. Both sides of each section were 
analyzed in order to increase the probability of identifying apoptotic neurons. Even though 
fewer total sections were analyzed than in the Motoneuron Number analysis, more total 
area was analyzed p)er animal since both the right and left side of each section was 
examined. 



29 



Statistics 

Analysis of variance, Fisher's protected least significant difference post-hoc test, 
and Student's t-test were performed using the StatView program (Abacus) on a Macintosh 
computer. 

Results 

Embryonic Observations 
Curai'e-ethanol coadministration study 

Survival varied greatly according to the treatment of each embryonic group. In the 
Curare group survival was approximately 47%, Saline group survival was 76%, 
Uninjected group survival was 96%, Ethanol group survival was 4%, and Curare+Ethanol 
group survival was 0.83%. This latter survival figure indicates that the combination of 
curare and ethanol was highly toxic to the developing chick embryo. The Curare embryos 
appeared to be more vascularized, in both the body and the limbs, than the controls. In 
contrast, the Ethanol and Curare+Ethanol embryos had wider bodies due to extensive 
bloating from the ethanol treatment. The spinal column in Curare+Ethanol embryos was 
softer than in the control counterparts and in the Ethanol embryos the lumbar region 
exhibited a minimal enlargement compared to Saline, Uninjected, and Curare embryos. 
The profound effects of ethanol treatment were verified by the presence of a green-colored 
liver in the Ethanol and Curare+Ethanol embryos, compared to the normal brown-colored 
liver in the remaining three groups. The alteration of the appearance of the liver in animals 
exposed to ethanol should be investigated further. The pathology of the liver could be 
analyzed and perhaps related to the death of embryos following ethanol exposure. 
Collaboration with a pathologist in examining this tissue would be preferable so that an 
accurate estimation of the method of liver damage can be determined. 



30 

Late exposure study 

Survival between experimental groups was not as variable in the Late Exposure 
study as it was in the Coadministration study. Approximately 60% of the Ethanol embryos 
and 75% of the Saline embryos survived. The livers of the Ethanol embryos were green, 
thus indicating that ethanol administration did have some general effect. The Saline 
embryos exhibited the normal brown liver coloration. The Ethanol embryos were not 
bloated in appearance, as has been observed in earlier studies (Heaton and Bradley, 1995) 
and the current Coadministration study, perhaps because the embryos of this age have the 
ability to clear ethanol from the bloodstream (Wilson et al., 1984). 
Motoneuron Counts 
Curare-ethanol coadministration study 

Analysis of variance indicated a significant effect due to treatment among these 
groups (F=23.061; df=28; p<0.0001). The number of motoneurons present in the lumbar 
region of the spinal cord in each of the five experimental groups at E12, could be described 
as one of three possible outcomes: average, above average, and below average. For these 
distinctions, "average" is defined to be a number of motoneurons similar to normal or 
control levels (i.e.. Saline or Uninjected); "above average" is defined to be a number of 
motoneurons significantly higher than average; and "below average" is defined to be a 
number of motoneurons significantly lower than average. Figure 2- 1 displays all of the 
groups utilized m this study. It should be noted that Figure 2-1 represents the number of 
motoneurons counted in each embryo and does not attempt to estimate the population of 
motoneurons in the lumbar spmal cord. Looking at Figure 2-1 from left to right shows 
how evident the three-tiered outcome of this study was. For example, the Curare group 
(located at the left in Figure 2-1) is the only group that displayed above average numbers. 
In fact, post hoc testing showed that the curare group contained significantly more 
motoneurons (about 35% more) than all other groups (p<0.0001 for each comparison). 
The Ethanol group (located at the right in Figure 2-1) was the only group to exhibit below 



31 



5 
U 

o 



4) 
Q 



3000 






2000- 



1000- 




Curare 

Curare+Ethanol 
n Uninjected 
■ Saline 

Ethanol 



Figure 2-1. Number of motoneurons in lumbar spinal cord at E12 following 
treatment from E4toEll. Motoneuron counts are displayed as means + SEM. 
Data from Curare, Curare+Ethanol, Uninjected, Saline, and Ethanol embryos are 
displayed, a = significantly more motoneurons than Curare+Ethanol, Uninjected, 
Saline, and Ethanol (p<0.0001 in all cases), b - significantly less than 
Curare+Ethanol, Uninjected, and Saline (p<0.005 in all cases). 



32 

average numbers. This fact is evidenced by the observation that post hoc testing indicated 
that the Ethanol group contained fewer motoneurons (about 20% fewer) than Saline, 
Unmjected, and Curare+Ethanol groups (p<0.005 in each comparison). All of the other 
groups-Saline, Uninjected, and Curare+Ethanol -contained average numbers of 
motoneurons and there were no significant differences among these groups. The 
Motoneuron Size and Spinal Cord Length Analyses section below suggests that 
motoneuron number differences observed in this study are not due to changes m the overall 
length of the spinal cord. 

Figure 2-2 is a collection of photomicrographs obtained from midlumbar segments 
of Saline, Ethanol, Curare, and Curare+Ethanol embryos. It is apparent that motoneuron 
number was affected by treatment as the Ethanol section contains the fewest motoneurons. 
The Saline section contains more than the Ethanol section, but not as many as the Curare 
section. Finally, the Curare+Ethanol section contains a similar number as the Saline 
section (refer to Figure 2-2). 
Pre-cell death exposure 

This analysis was undertaken to determine if ethanol exposure prior to the period 
for NOCD (E4 to E5) in the spinal cord could affect overall motoneuron number at E12. 
The T-test indicated no significant difference in the number of motoneurons in the lumbar 
spinal cord between the Ethanol and Saline embryos. This result suggests that exposure to 
ethanol before administration of curare, and NOCD, had no adverse effect on the 
motoneuron population of the lumbar spinal cord. Therefore, results obtained following 
ethanol exposure from E4 to El 1 are not confounded by the fact that ethanol exposure did 
not coincide completely with the period for NOCD. 
Late exposure study. 

The results obtained following late ethanol exposure were similar to those obtained 
when ethanol was administered from E4 to Ell, in that Ethanol embryos exposed from 
ElO to E16 exhibited a significant reduction in motoneuron number. The length of each 



Figure 2-2. Photomicrographs of coronal sections from the midlumbar region of E12 
spinal cords. A. Ethanol, B. Saline, C. Curare, and D. Curare+Ethanol spinal cords. 
Note that the Curare "bulge" is most pronounced while the Ethanol "bulge" is hardly 
apparent. Also note the density of motoneurons in the lateral motor column. The Curare 
contains more and more densely packed motoneurons, while the Ethanol cord contains 
fewer and less densely packed motoneurons. The Saline and Curare+Ethanol spinal cord 
sections contain roughly similar numbers of motoneurons, n = 6 Ethanol, 10 Saline, 6 
Curare, and 5 Curare+Ethanol 



34 











Saline 



Ethanol 







Curare 



Curare+Ethanol 



Figure 2-2. 



35 

embryo's spinal cord was similar since similar numbers of sections were counted from 
each embryo and each section is 12;^m thick (see section below). The t-test indicated that 
the E16 Ethanol embryos contained significantly fewer motoneurons (approximately 15%) 
than did their Saline counterparts (p<0.05). Figure 2-3 displays the data generated in this 
portion of the study. 

Motoneuron Size and Spinal Cord Length Analyses 
Curare-ethanol coadministration study 

Analysis of variance of the motoneuron size data indicted a significant effect due to 
treatment (F=3.233; df=200; p<0.05). The only significant difference indicated by post 
hoc testing was that the Curare+Ethanol group contained significantly smaller motoneurons 
than the Saline (p<0.005), Ethanol (p<0.005), and Curare (p<0.05) groups. There were 
no other significant differences between any of the other groups. Analysis of variance of 
spinal cord length found no significant effect due to treatment. Table 2-1 shows the data 
generated in this portion of the study. Note that even though the difference between the 
Curare+Ethanol group and the other groups is less than 2 pim, it is significant. 



Table 2-1. Cell size and spinal cord length. 

Coadministration Study 

Group Cell Size (pim) 



Spinal Cord Length (/^m) 



Ethanol 


19.025 + 0.404 


3940.0 + 95.04 


Saline 


18.875 + 0.399 


3750.0 + 76.92 


Uninjected 


18.200 + 0.431 


3614.9+151.80 


Curare 


18.725 + 0.410 


3800.8+119.20 


Curare+Ethanol 


17.275 + 0.326 * 


3993.2 + 68.76 


Late Exposure Stu 

Group 


idy 

Cell Size (pim) 


Spinal Cord Length (pim) 


Ethanol 


20.950 + 0.399 


6000.0 + 138.6 


Saline 


20.650 + 0.408 


6060.0 + 187.8 



All values are means + SEM. For cell size, n=40 for each group. For spinal cord length 

Ethanol n=6, Saline n=10, Uninjected n=6, Curare n=6, and Curare+Ethanol n=5. 

* denotes significance in comparison to Ethanol (p<0.005), Saline (p<0.005), and Curare 

(p<0.05). 



36 



^3 

I 

O 

U 

I 

O 



o 
I 



2000 



1000 




Saline 



Ethanol 



Figure 2-3. Number of motoneurons in lumbar spinal cord at El 6 following 
ethanol treatment from ElO to E15. Motoneuron counts obtained in the Late 
Exposure study are displayed as means + SEM. Data from Ethanol and Saline 
embryos are displayed, a = significantly fewer motoneurons than Saline 
(p<0.05). 



37 



Late exposure study 

The t-test indicated that there was no difference in motoneuron size or spinal cord 
length between the Ethanol and Saline groups. Table 2- 1 also contains data generated from 
this portion of the experiment. 

Crude Muscle Extract Study 

As mentioned above, both neuronal survival and neurite outgrowth at 24 and 48 
hours were observed in this portion of the study. Also recall that muscle tissue was 
prepared from E16 embryos exposed to ethanol from ElO to E15. Analysis of variance 
indicated no significant effect due to treatment for survival at either 24 or 48 hours. 
Likewise, after 24 hours in culture there was no significant effect due to treatment for 
outgrowth. Following 48 hours in culture, however, there was a significant effect of 
treatment for outgrowth (F=4.232; df=18; p<0.05). Post-hoc testing revealed that Ethanol 
cultures extended significantly more neurites than NC cultures (p<0.05). However, 
Ethanol extract and Saline extract cultures were not different, thus this result does not 
indicate that ethanol increases neurotrophic activity in chick limb muscle. Neurite 
outgrowth at 48 hours in Saline cultures approached statistical significance (p=0.078), 
which further supports the notion that observations obtained from Ethanol and Saline 
extract cultures were similar. Table 2-2 displays the results obtained in this portion of the 
study. 

Table 2-2. Neurotrophic activity of crude muscle extract. 



Group 



Survival (% of 
initial counts) 

24 Hours 48 Hours 



Outgrowth (% of 
surviving cells) 

24 Hours 48 Hours 



Negative Control 


83.486+2.861 


77.929+3.288 


9.143+1.788 


12.243+2.095 


Saline 


86.314+1.766 


81.443+1.918 


11.971+2.171 


17.557+1.547 


Ethanol 


88.614+1.840 


84.529+1.526 


12.400+2.530 


20.386+2.309 * 



All values are means + SEM. n=8 (one culture produced from each of eight embiyos) for 
all groups. * denotes significance in comparison to Negative Control (p<0.05). 



38 

Assessment of Apoptotic Cells 

As mentioned above, spinal cords for intermediate time points in the I^te Exposure 
Study (E12 and E14) were assessed for apoptotic cells. Three embryos from the Saline 
group and three embryos from the Ethanol group were studied as described above. At 
E12, a total of 2 apoptotic cells were identified among the animals in the Saline group and a 
total of 3 apoptotic cells were identified among the Ethanol group. As stated above, an 
average of 21 sections— and both sides of each section-for each animal were examined. 
Since a total of 5 apoptotic cells were identified at El 2, it is unlikely that an analysis of 
more sections would result in finding significantly more apoptotic motoneurons. Both 
sides of each section were analyzed for apoptotic cells so that a greater total area was 
analyzed in this portion of the study than in the Motoneuron Counts section. At E14, a 
total of 2 apoptotic cells were identified among the animals in the Saline group and a total of 
4 apoptotic cells were identified from the Ethanol group (an average of 30 sections were 
examined in each E14 embryo). These results led to the conclusion that there was no 
significant difference between the Ethanol and Saline groups in numbers of apoptotic cells 
when ethanol was administered from ElO to E15. However, the data do not conclusively 
eliminate apoptosis as a potential mechanism for ethanol in this neuronal population. This 
possibility will be discussed further below. 

Discussion 

Curare-Ethanol Coadministration Study 

The results of the Curare-Ethanol Coadministration study indicate that ethanol is 
toxic to developing motoneurons even in the absence of NOCD. This result suggests that 
ethanol does exhibit a mechanism other than exacerbation of NOCD to developing 
motoneurons and the change in motoneurons number is not due to a change in spinal cord 
length. The Curare embryos had significantly more motoneurons than all other groups. 
This result agrees with prior results from other laboratories (Pittman and Oppenheim, 1 979; 
Pittman and Oppenheim, 1978). The mechanism for curare rescue of motoneurons 



39 

destined to die of NOCD has been studied previously. Curare increases the number of 
acetylchohne receptor clusters in developing myofibers (Oppenheim et al., 1989). The 
implication of this result is that greater numbers of synapses can form and developing 
motoneurons would then have greater access to target derived NTFs (Oppenheim, 1991). 
The fact that the Curare+Ethanol group contained significantly fewer motoneurons than the 
Curare group indicates that ethanol does not reduce motoneuron number by exacerbating 
NOCD. Since curare administration suspends NOCD, ethanol must cause additional 
motoneurons to perish by a mechanism other than exacerbation of NOCD. Potential 
mechanisms for ethanol toxicity are discussed further below. The three-tiered level of 
motoneuron survival observed in this study also suggests that ethanol and NOCD act 
independently. The Curare group, which contains that largest number of motoneurons, is 
not subject to either cell death process. The middle tier contains three groups that are all 
subject to one of the two cell death processes: Uninjected (NOCD), Saline (NOCD), and 
Curare+Ethanol (ethanol toxicity). The Ethanol group, which contained the fewest number 
of motoneurons, was subject to both cell death processes and exhibited an additive effect of 
motoneuron loss (refer to Figure 2-1). 

The timing of ethanol and curare administration for this experiment was designed to 
coordinate the presence of curare with the onset of NOCD in the lumbar spinal cord. The 
results indicated that ethanol exposure on E4 and E5 did not reduce motoneuron number. 
There are two implications of this result: Ethanol exposure prior to curare administration 
had no adverse effect on the motoneuron population of the lumbar spinal cord and the 
critical period for ethanol -induced motoneuron death does not include E4 and E5. A 
possible addition to this study would be a study which further limits exposure time to 
ethanol and attempts to define a critical period where ethanol exerts its greatest toxic effects 
on this neuronal population. 



40 



Late Exposure Study 

The results of the Late Exposure study indicate that ethanol has the ability to reduce 
motoneuron number in the lumbar section of the spinal cord during a later period of 
development. Since the reduction in motoneuron number during this late exposure period 
does mimic the reduction observed following ethanol administration from E4 to Ell, it is 
logical to assume that ethanol might have the ability to reduce motoneuron number in the 
developing chick embryo during any time period of motoneuron development and perhaps 
during later periods as well. This result is further evidence that ethanol is directly toxic to 
motoneurons of the embryonic chick because the ethanol exposure took place following the 
period of NOCD. Additionally, since spinal cord length was unaltered following ethanol 
treatment, this study suggests that these findings are not an artifact of a change in spinal 
cord volume. 

Additional experiments were conducted to determine whether ethanol exposure 
during this late period altered the neurotrophic activity of embryonic chick muscle. In a 
previous study, this laboratory found that neurotrophic activity of chick muscle from limb 
bud was reduced following ethanol exposure from E4 to E8 (Heaton and Bradley, 1995). 
However, the current results suggest that there is no difference in muscle neurotrophic 
activity in embryos treated with ethanol or saline. The results suggest that ethanol does not 
reduce motoneuron number by decreasing the total amount of neurotrophic support 
available to the motoneuron population as it does when administered during the earlier stage 
of development (Heaton and Bradley, 1995). However, it does remain a possibility that 
individual NTFs produced by muscle are altered in their expression such that one factor 
was upregulated while another was downregulated. Such a change could alter survival of 
the NTF dependent motoneuron population if the downregulated factor was critical for 
survival at the given time period of ethanol administration. 



41 



General Discussion 

The results of the present study offer many possibilities for the action of ethanol in 
the developing motor system. Since our analyses included motoneuron survival, 
neurotrophic activity, and apoptotic cells, this study has the ability to question potential 
mechanisms of ethanol toxicity that are suggested by previous research. Specific areas, as 
they relate to ethanol toxicity in the developing nervous system, are discussed below. 
Disruption of NTF support 

Ethanol has been shown to affect neurotrophic activity and neuronal responsiveness 
to NTFs (Heaton et al., 1995b; Heaton et al., 1992; Heaton et al., 1993; Heaton et al., 
1994). Since developing motoneurons of the lateral column require neurotrophic support 
for survival (Oppenheim, 1991), it is possible that ethanol may interfere with the ability of 
these cells to gain access to NTFs and therefore cause excess cell death. The results of the 
current experiments are somewhat contradictory. As was mentioned above, extracts made 
from E16 leg muscle following ethanol exposure from ElO to E15 were significantly 
increased in neurotrophic activity in comparison to NC cultures. NC cultures were 
composed of cell cultures grown in regular culture medium, with essentially little 
exogenous neurotrophic support. Thus, these cultures were negative control groups. Only 
Saline and Ethanol cultures contained exogenous neurotrophic support. Since NTF 
activity— as measured by both neurite outgrowth and survival— was not significantly 
increased in comparison to the positive control group (Saline), it would be erroneous to 
conclude that ethanol exposure increases neurotrophic activity of developing leg muscle. 
The current study suggests that late ethanol exposure does not affect the neurotrophic 
content of chicle limb muscle— a result that contrasts with earlier findings that demonstrated 
ethanol exposure from E4-E8 reduces neurotrophic content of developing limb muscle 
(Heaton and Bradley, 1995). 

The reason for this disparity may lie in the fact that neurotrophic content of 
developing limb muscle increases throughout development and peaks at E18 (Thompson 



42 

and Thompson, 1988). The fact that total neurotrophic activity is nearing its peak at E16 
suggests that overall activity is much higher at E16 than at E8. Any gross change in 
neurotrophic activity at E8 would result in a larger percentage change in activity than a 
similar change at E16. The implication would be that enough residual neurotrophic activity 
would remain in E16 muscle to continue to support the spinal cord cultures, whereas the 
support would be reduced sufficiently to alter the growth of the cultures when ethanol was 
administered earlier in development. The fact that Saline embryos did not significantly 
increase neurotrophic activity in comparison to the NC group should be discussed further. 
The answer may lie in the ontogeny of neurotrophic activity in developing limb muscle. 
Since this level increases until reaching a peak at E18 (Thompson and Thompson, 1988), 
the relative amount of neurotrophic factors present in extract is increased in comparison to 
extracts prepared on E8. Since high levels of NTFs in cultures can be lethal, it is possible 
that the amount of trophic activity released into the culture medium ceased to be supportive. 
As was mentioned above, the amount of gross protein, not gross neurotrophic activity, was 
regulated in these cultures. Therefore, since NTFs are increased relative to overall protein 
level (Thompson and Thompson, 1988), our cultures may not have been maintained for 
peak neurotrophic activity. 

Another possible explanation for the fact that Saline cultures did not exhibit 
significantly greater neurotrophic activity in comparison to the NC is the fact that FBS was 
used in the culture medium. Since FBS contains NTFs as well as other undefined proteins, 
it is likely that the NC cultures exhibit growth and survival far above levels that would be 
present without FBS. Clearly, further experimentation should be completed before 
concluding that ElO to E15 ethanol exposure in embryonic chick increases neurotrophic 
activity of developing leg muscle. Trophic support for developing motoneurons is not 
limited to, but is in a large part provided by, target muscle. Glia in the spinal cord produce 
NTFs that support developing motoneurons (Arce et al, 1998). However, motoneuron 
number in the chick has been shown to be regulated by the amount of target muscle 



43 

present. Specifically, when a limb is removed from a chick embryo, motoneuron number 
is reduced accordingly (Caldero et al., 1998; Lanser and Fallon, 1987). When a 
supernumerary limb is grafted onto an embryo, motoneuron number is increased (HoUyday 
and Hamburger, 1976). Thus, target muscle regulates motoneuron number in a "dose- 
dependent" manner. This relationship has been further confinned by the removal of 
varying portions of limb bud from developing chick embryos. In this case the survival of 
motoneurons was proportional to the amount of limb bud remaining (Lanser and Fallon, 
1987). 
Response to NTFs 

This study found that ethanol did not significantly alter the gross amount of 
neurotrophic activity produced in target limb muscle in comparison to Saline treated 
embryos when administered from ElO to E15. The significantly greater neurite outgrowth 
of the Ethanol group in comparison to the NC group does not provide conclusive evidence 
that embryonic ethanol exposure increases neurotrophic activity. When this result is 
combined with the fact the ethanol reduces motoneuron number in the spinal cord during 
this period, it could be that ethanol has altered the ability of motoneurons to respond to 
neurotrophic support produced by the target muscle. This change in the ability to respond 
to NTFs could be achieved by altering retrograde transport capacity, receptor expression, 
or receptor function. Ethanol is known to hinder retrograde transport in cultured 
thymocytes (McLane, 1990) and previous research in this laboratory found that prenatal 
exposure to ethanol in the rat reduced the ability of cultured hippocampal neurons to 
respond to exogenous NTFs (Heaton et al., 1994). Such a mechanism might occur by 
ethanol altering expression of NTF receptor genes or by altering the activity of the active 
receptor. 

Ethanol is known to affect certain receptor systems. For example, N-Methyl-D- 
aspartate (NMDA) receptors are a type of glutamate receptor and are involved in long-term 
potentiation, which has long been thought to be involved in how the hippocampus encodes 



44 

new memories (Bunsey and Eichenbaum, 1996). Ethanol has been shown to inhibit the 
flow of ions through NMDA receptors and to block NMDA receptor antagonists from 
binding to the receptor (Lovmger et al, 1989; Valles et al., 1995). Previous research from 
this laboratory has implied that ethanol inhibits neuronal ability to respond to NTFs. 
Specifically, bFGF's ability to promote neurite outgrowth in hippocampal cultures was 
reduced in those composed of cell from animals exposed prenatally to ethanol (Heaton et 
al., 1995b). Such a mechanism, inhibition of the NTF/receptor binding system, could be 
involved in ethanol 's toxic effect upon the neuromuscular system. Future experiments will 
be designed to determine whether such a mechanism is indeed occurring in this system. 

Since the neurotrophic content of El 6 muscle exposed to ethanol from ElO to E15 
is not significantly changed in comparison to Saline exposed embryos, it is likely that 
ethanol interferes with an individual motoneuron's ability to utilize its neurotrophic support 
rather than reducing neurotrophic support. In support of this hypothesis, our lab has also 
found that ethanol disrupts the ability of co-cultures of spinal cord to grow neurites toward 
limb muscle tissue (Heaton et al., 1995a). This result could also be due to altered NTF 
receptor function since neurite outgrowth occurs as the growth cone responds to its 
environment. If the ability of the growth cone to sense its environment were diminished, it 
would not extend the neurite in a normal manner. 

The fact that motoneuron and muscle development proceed concurrently and are 
interdependent should not be overlooked. Ethanol does reduce the amount of trophic 
substances produced in muscle when administered early in embryonic development 
(Heaton and Bradley, 1995) and this further limitation of trophic factors could cause fewer 
motoneurons than normal to survive. The current results do not support such a hypothesis 
during late ethanol exposure since ethanol administered from ElO to E15 did not reduce or 
otherwise alter neurotrophic activity of limb muscle extracts in comparison to the positive 
control group. Saline. Motoneuron number is reduced following both exposure periods 
which suggests that NTF developmental activity is not solely responsible for this loss in the 



45 

embryonic chick, at least during the late exposure period. Since muscle requires 
motoneuron innervation and activity to develop properly (Ishiura et al., 1981), it is equally 
possible that a directly toxic effect of ethanol on motoneurons, such as a change in the 
ability of motoneurons to respond to NTFs as mentioned above, could cause developing 
muscles to exhibit the malformations noted in both human and animal models of prenatal 
ethanol exposure (Adickes and Shuman, 1983; Nyquist-Battie et al., 1987). 
Apoptosis 

While the current experiments have been effective in eliminating exacerbation of 
NOCD as a potential mechanism of ethanol toxicity observed in the developing chick 
embryo spinal cord, other possible explanations for ethanol toxicity exist. These potential 
mechanisms could utilize an apoptotic mechanism to achieve cell death. NOCD has been 
shown to be an apoptotic process that requires the cell to participate actively in its own 
demise (Columbano, 1995). The term "active" in this context indicates that new RNA and 
protein synthesis are required for apoptosis to proceed. In fact, NOCD in the spinal cord 
does require new RNA and protein synthesis (Oppenheim et al., 1989), but some examples 
of apoptosis in the absence of RNA synthesis have been documented (Kelley et al., 1992). 
In culture, ethanol has been shown to induce apoptosis in hypothalamic neurons (De et al., 
1994), thymocytes (Ewald and Shao, 1993), and cerebellar granule neurons (Bhave and 
Hoffman, 1997; Liesi, 1997). Previous studies have also implicated chronic ethanol 
treatment in producing apoptotic cell death in the hippocampus and the cerebellum of adult 
rats in vivo (Renis et al., 1996; Singh et al., 1995). Additionally, there is evidence that 
ethanol increases programmed cell death in the cerebellum (Cragg and Phillips, 1985). 

The present study did attempt to determine whether ethanol induced apoptosis in 
motoneurons of the lumbar spinal cord. However, the results did not indicate that such a 
mechanism was occurring. Saline and Ethanol group spinal cords that were examined at 
E12 and E14 contained virtually identical, and very few, numbers of motoneurons 
undergoing apoptosis identified by histological or morphological characteristics. The fact 



46 

that such cells were not seen does not rule out the possibility that ethanol is inducing 
apoptosis in spinal cord motoneurons but was not detected with this methodology. As was 
mentioned above, ethanol exposure from ElO to E15 results in a reduction of 
approximately 15% in motoneuron number in the lumbar spinal cord. Furthermore, 
apoptosis is a relatively rapid cellular process that is completed in approximately 3 hours 
(Bursch et al., 1990) and begins very soon after ethanol exposure (Cragg and Phillips, 
1985). To have the best opportunity to observe apoptosis, embryos should have been 
sacrificed between two and five hours following the ethanol injection on days when this 
analysis was to occur. 
Hypoxia 

Another possible mechanism contributing to the toxic effects of ethanol upon 
developing motoneurons is hypoxia. Hypoxia, a condition which results from inadequate 
blood supply, exerts a variety of effects on all organ systems, including the central nervous 
system (CNS). Previous studies in mammals have found that administration of ethanol 
causes a decrease in umbilical artery blood flow and a reduction of oxygen delivery (Altura 
et al., 1983; Jones et al., 1981; Mukherjee and Hodgen, 1982). However, a link between 
ethanol and hypoxia in the developing chick has not been established. Hypoxia alone has 
been studied in this animal model and has been shown to reduce the overall vascularity of 
the chorioallantoic membrane (Strick et al., 1991) and reduce its blood flow (Ar et al., 
1991). The fact that the direct relationship between ethanol and hypoxia has not been 
explored in the chick does not eliminate hypoxia as a potential mechanism in this model. A 
future goal of this laboratory should be to determine whether or not this relationship exists 
in developing chick embryos. In the CNS, hypoxic conditions affect hippocampal CAl 
pyramidal neurons and cerebellar Purkinje cells (Auer et al., 1989; Jorgensen and Diemer, 
1982). These same neuronal populations are damaged when rats are exposed to ethanol 
prenatally and postnatally (Barnes and Walker, 1981; Bonthius and West, 1990; Phillips 
and Cragg, 1982; Pierce et al., 1989). These studies have led to a hypothesis that ethanol- 



47 

induced hypoxia may cause excitotoxic damage to developing neurons (Michaelis, 1990). 

If such a mechanism were occurring in response to prenatal ethanol exposure, one would 

expect to find that ethanol has an effect on Ca^"" homeostasis. In fact, ethanol has been 

shown to regulate Ca^"^ homeostasis m cultured neurons (Koike and Tanaka, 1991; Webb et 

al. , 1995). These latter studies do suggest that hypoxia may be involved in ethanol 

toxicity. 

Additional considerations 

The motoneuron size findings indicate that for the most part ethanol has no effect on 
this aspect of motoneuron morphology in the lumbar spinal cord. The fact that 
Curare+Ethanol embryos contained smaller motoneurons could be due to the combined 
effect of curare and ethanol which were extremely toxic to the developing embryos. Since 
survival was poor in this group, it is likely that some general status of the embryo was 
compromised which could have altered motoneuron size. The finding that ethanol reduces 
motoneuron number at more than one stage of development suggests that it may have the 
ability to be toxic to motoneurons throughout chick nervous system development. 

The present study found that motoneuron number is reduced following ethanol 
administration from E4 to El 1 and from ElO to E15. This result is similar to results 
obtained from other neuronal populations such as cerebellar Purkinje cells which are 
susceptible to ethanol during a range of time periods (Hamre and West, 1993; Phillips and 
Cragg, 1982). Specifically, Purkinje cells are reduced in number following both prenatal 
(Phillips and Cragg, 1982), and postnatal (Hamre and West, 1993; Phillips and Cragg, 
1982) ethanol exposure. The similarity in temporal vulnerability between motoneurons and 
Purkinje cells suggests that some fundamental resemblance between these two populations 
determines their similar susceptibility to ethanol. To thoroughly and properly investigate 
the role of ethanol in motoneuron reduction in the chick lumbar spinal cord, completion of a 
parametric ethanol exposure study will be necessary. Such a study would allow us to 
determine whether ethanol is directly toxic throughout chick embryonic development or if 



48 

ethanol is toxic at multiple critical periods. This knowledge would then allow us to better 
hypothesize mechanisms for ethanol toxicity in this neuronal population. 



CHAPTERS 

CHARACTERIZATION OF MOTONEURON SURVIVAL FOLLOWING ETHANOL 

EXPOSURE AND CONCURRENT TREATMENT WITH EXOGENOUS GDNF OR 

BDNF IN THE EMBRYONIC CHICK SPINAL CORD 

Summary 

Maternal consumption of ethanol is widely recognized as a leading cause of mental 
and physical deficits. Many populations of the central nervous system (CNS) are affected 
by the teratogenic effects of ethanol. Neuroprotection against ethanol has been studied 
extensively in cell culture models and has also been studied in vivo in response to a variety 
of neurotoxic events including hypoxia, ischemia, and hypoglycemia. Some neurotrophic 
factors (NTFs) have been shown to protect against ethanol neurotoxicity in culture. The 
only in vivo evidence of NTF prevention of ethanol neurotoxicity involved NGF protection 
of choline acetyl transferase activity in early chick embryos (Brodie et al., 1991). Previous 
studies in this laboratory have demonstrated that ethanol is toxic to developing chick 
embryo motoneurons when administered from embryonic day 10 (ElO) to E15. Other 
laboratories have found that developing motoneurons are dependent on glial cell line- 
derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). 
GDNF and BDNF suspend naturally occurring cell death (NOCD) in a subset of 
developing motoneurons. These factors also rescue motoneurons from axotomy-induced 
cell death in developing embryos. The concurrent delivery of GDNF with ethanol and 
BDNF with ethanol was designed to lest their ability to provide neuroprotection for this 
ethanol -sensitive motoneuron population. Analysis of motoneuron number indicated that 
GDNF, but not BDNF, significantly increased motoneuron number in the developing 
spinal cord following embryonic ethanol exposure. However, GDNF was not found to 



49 



50 

interact significantly with ethanol. Therefore, GDNF may serve to increase motoneuron 
number to a level that is significantly greater than in ethanol treated embryos by a 
mechanism that is independent of ethanol. Further studies should be developed to examine 
this phenomenon in greater detail and determine whether GDNF does indeed provide 
protection from ethanol toxicity. 

Introduction 

As was mentioned previously, much evidence has been gathered regarding the 
effects ethanol exerts in the developing nervous system (Barnes and Walker, 1981; Jones 
and Smith, 1973; Miller, 1986; Streissguth et al., 1991; West, 1986). Fetal alcohol 
syndrome (FAS) produces CNS deficits that do not lessen as the patient ages (Streissguth, 
1993). Postmortem analysis of human FAS neuropathology has identified CNS 
abnormalities which include disorders of laminae of the cerebral cortex, cerebellar 
abnormalities, a reduction of dendritic spines on cortical pyramidal cells, hippocampal 
malformation, and microcephaly (Claixen et al., 1978; Ferrer and Galofre, 1987). 
Neuronal populations that are known to be affected by ethanol in animal models include the 
cerebellum (Cragg and Phillips, 1985; West, 1986), the septohippocampal system (Barnes 
and Walker, 1981), cerebral cortex (Miller, 1986), the substantia nigra (Shetty et al., 
1993), chief sensory trigeminal nucleus (Miller and Muller, 1989), red nucleus (Zajacet 
al., 1989), inferior olivary nucleus (Napper and West, 1995), striatum (Heaton et al., 
1996) and motoneurons of the spinal cord (Bradley et al., 1997; Heaton and Bradley, 
1995). Some of the microscopic and molecular changes that have been observed following 
ethanol exposure include decreased dendritic arborization (Davies and Smith, 1981), 
delayed synaptogenesis (Leonard, 1987), decreased neurotransmitter synthesis (Rawat, 
1977; Swanson et al., 1994), changes in connectivity (West et al., 1994), and cell loss 
(Barnes and Walker, 1981 ; Bauer-Moffet and Altman, 1975; West et al., 1986). 

Ethanol affects chick embryo development in a manner similar to mammals. As 
was mentioned previously, chicks exposed to ethanol prenatally have been shown to 



51 

exhibit reduced brain size, brain weight, DNA and protein synthesis (Pennington and 
Kalmus, 1987), and reduced neurotransmitter synthesis (Brodie and Vemadakis, 1990; 
Swanson et al., 1994). An advantage of using a chick model to study ethanol is the fact 
that ethanol can be administered in exact doses to the developing embryo, and only 
molecules produced by the embryo itself can remove the ethanol from the embryonic 
environment. Maternal influences are removed when using the chick embryo as ethanol is 
cleared from the bloodstream by the mother in a mammalian system. In the chick , the 
embryo is isolated as it develops. The chick model allows the investigator to observe direct 
effects of ethanol without possible interactions of maternal metabolism interfering. While 
chick development is clearly different from mammalian gestation (and this is a caveat of 
using the chick embiyo as a model for FAS), this model allows researchers to study in vivo 
interactions in a developing organism that are not possible in a mammalian model. This 
model has been widely used to study the effects of NTFs on the developmg motor system 
(Oppenheim et al., 1995; Oppenheim et al., 1992). NTFs can be administered through 
windows in the egg shell directly onto the chorioallantoic membrane. Developing chick 
embryos tolerate slight invasions into their environment as long as the underlying 
membranes are not disrupted. 

The present studies focus on the motor system of the developing chick embryo. 
Motoneurons have been shown to be susceptible to the toxic effects of ethanol both in 
culture (Dow and Riopelle, 1985; Heaton et al., 1995b) and in vivo (Bradley et al., 1997; 
Heaton and Bradley, 1995; Heaton et al., 1995b). Our laboratory has found that ethanol 
can reduce motoneuron number when administered to chick embryos from E4 to El 1 
(Heaton and Bradley, 1995) and when administered from ElO to E15 (Bradley et al, 
1997). This reduction is not dependent on NOCD since curare, an agent which blocks 
NOCD, does not prevent this ethanol-induced death (Bradley et al., 1997). Furthermore, 
when the period for NOCD had expired, ethanol still reduced motoneuron number (Bradley 
etal., 1997). 



52 

Two NTFs important for motoneuron development are BDNF and GDNF. BDNF 
is a member of the neurotrophin family of NTFs which includes nerve growth factor 
(NGF), neurotrophin-3, and neurotrophin-4/5. A 118 amino acid residue polypeptide (Hag 
et al., 1994), BDNF forms homodimers to attain its active form and binds with high 
affinity to tyrosine receptor kinase B (trkB; Klein et al., 1991). p75 is the low-affinity 
binding receptor for all neurotrophms. The role of p75 in mediating neurotrophin-trk 
binding is unclear. Primary sensory neurons display no biological activity when nerve 
growth factor binds trkA in the absence of p75 (Verge et al, 1992), while in other cell 
types the trk receptors can work alone (Klein et al., 1991). BDNF is produced by the 
developing skeletal muscle and is known to support motoneuron survival during 
development and to protect motoneurons of both the chick and rat from degenerating after 
lesion (Oppenheim et al., 1992; Sendtner et al, 1992; Yan et al., 1992). In addition to this 
expression, BDNF is expressed in the hippocampus, adrenal gland, and in whole brain 
during rat development (Maisonpierre et al, 1990). In the rat, mRNA for BDNF is 
expressed in skeletal muscle both prenatally and postnatally (Griesbeck et al, 1995). 

GDNF is a member of the transforming growth factor B (TGF-B) superfamily and 
naturally occurs as a dimer with a molecular weight of 40-45 kD (each molecule 134 amino 
acid residues; Lin et al, 1993). Recent studies suggest that GDNF and its receptors, 
GDNFRoc and c-ret, form a complex that allows c-ret to transduce the signals from GDNF 
(Jing et al., 1996; Treanor et al, 1996). In this complex, GDNF'Roc acts as a ligand- 
binding protein by binding GDNF (Jing et al., 1996). The GDNFR^-GDNF complex 
then forms a complex with c-ret (Jing et al., 1996; Treanor et al., 1996). C-ret is the only 
molecule of the complex capable of producing intracellular signals (Rosenthal, 1997). 
During embryogenesis of the rat, GDNF niRNA is expressed by mesenchymal cells and in 
developing skeletal muscle beginning at E15, and in developing skin beginning at E17 
(Nosrat et al, 1996; Trupp et al, 1995; Wright and Snider, 1996). GDNF is also 
expressed peripherally in the teeth, tongue, retina, nasal cavity, ear, kidney, and 



53 

gastrointestinal tract during various stages of development (Nosrat et al., 1996). In the 
CNS, GDNF is expressed in the striatum, hippocampus, and cerebellum beginning at E15, 
and in the trigeminal motor nucleus (E17) and cortex (postnatal day 7). C-ret is highly 
expressed in substantia nigra dopaminergic neurons, a population which is protected from 
6-hydroxydopamine (6-OHDA) lesion by exogenous GDNF (Trupp et al, 1995). Other 
populations that are responsive to GDNF express c-ret, including spinal motoneurons 
(Pachnis et al., 1993; Tsuzuki et al., 1995) and certain subpopulations of the peripheral 
ganglia (Pachnis et al., 1993; Tsuzuki et al., 1995). 

Studies of genetically altered mice, where a specific gene has been deleted from the 
genome, have added to the knowledge of NTFs and their receptors. Knockout mice have 
been created to study the relative importance of BDNF, trkB, p75, GDNF, and c-ret. 
Since the present study is concerned with the development of the motor system, discussion 
of these animals will be limited to this subject. While BDNF and p75 knockout mice do 
not display a loss of motoneurons (Jones et al., 1994; Lee et al., 1992), trkB knockout 
mice do contain reduced motoneuron number (Klein et al., 1993). The situation with 
regard to GDNF and its receptor is somewhat different. GDNF deficient animals exhibit a 
small but significant loss of motoneurons (Moore et al., 1996), while c-ret knockout mice 
do not exhibit reduced numbers (Marcos and Pachnis, 1996). These results suggest that 
the receptors that actually transduce the signals of BDNF and GDNF are important for 
proper motoneuron development. Additionally, these results suggest that there may be 
some redundancy in the NTFs that can activate trkB and c-ret, since GDNF-deficient and 
BDNF-deficient animals exhibit no deficit in motoneuron number. 

Neuroprotection by polypeptide growth factors has been studied extensively in 
recent years. Examples of neuroprotection include epidermal growth factor protection of 
whole brain neuronal cultures from anoxia (Pauwels et al., 1989), NGF protection of rat 
hippocampal and human cortical neurons from hypoglycemia (Cheng and Mattson, 1991), 
and basic fibroblast growth factor (bFGF) prevention of thalamic degeneration following 



54 

cortical infarction (Yamada et al., 1991). In the developing nervous system, GDNF has 
been shown to be potent in protecting neurons from a variety of conditions that normally 
cause death such as NOCD (Oppenheim et al., 1995), 6-OHDA lesion (Choi-Lundberg et 
al., 1997; Keams and Gash, 1995; Tomac et al., 1995), and axotomy (Gimenez y Ribotta 
et al., 1997; fiouenou et al., 1996; Oppenheim et al., 1995). BDNF is also effective in 
providing neuroprotection from toxic events. For example, BDNF rescues some neurons 
ischemia-induced cell death in rat hippocampal slice cultures (Pringle et al., 1996). BDNF 
has also been shown to reduce NOCD in motoneurons (Oppenheim et al., 1992) and 
apoptotic death in PC12 cells (Jian et al., 1996) and cultured rat cerebellar granule neurons 
(Kubo et al, 1995). The fact that both GDNF and BDNF provide such potent support for 
developing and injured neurons suggests that both could protect motoneurons from 
ethanol-induced death. 

Neuroprotection from ethanol has been demonstrated in culture in previous 
research. Previous studies in our laboratory found that NGF can protect cultured dorsal 
root ganglion (DRG) neurons (Heaton et al., 1993) and septal neurons (Heaton et al., 
1994) from ethanol toxicity. bFGF was shown to afford some neuroprotection to cultured 
septal and hippocampal neurons (Heaton et al., 1994). NGF and bFGF protect cultured 
cerebellar granule cells from ethanol-induced cell death (Luo et al., 1997). This 
neuroprotection afforded by NGF and bFGF was found to require both protein and RNA 
synthesis (Luo et al., 1997). This result suggests that neuroprotection is related to a signal 
that the NTF receptor sends to the nucleus of the cell. NGF protection of choline acetyl 
transferase activity from ethanol was the first in vivo demonstration of NTF 
neuroprotection from ethanol (Brodie et al., 1991). The previous use of the chick embryo 
in both in vivo NTF (Oppenheim et al., 1995; Oppenheim et al., 1992) and ethanol 
research (Heaton and Bradley, 1995) makes it an excellent choice for studying ethanol- 
NTF interactions. 



55 

The objective of the present experiment was to determine whether exogenous NTFs 
could provide protection for motoneurons exposed to ethanol from ElO through E15. Both 
GDNF and BDNF are known to be NTFs for developing motoneurons (Henderson et al., 
1994; Oppenheim et al., 1995; Oppenheim et al, 1992; Sendtner et al., 1992; Yan et al., 
1992). The concurrent delivery of each of these NTFs with ethanol was designed to test 
the ability of each to provide neuroprotection for this ethanol-sensitive population. 
Analysis of motoneuron number indicated that GDNF, but not BDNF, resulted in 
increasing the number of motoneurons present in the lumbar spinal cord following ethanol 
exposure in the developing lumbar spinal cord. 

Materials and Methods 

Subjects 

White Leghorn chick eggs were obtained from the University of Florida Poultry 
Science Department. Eggs were placed in a Marsh incubator and maintained at 37°C and 
70% relative humidity until E4. At that time, the eggs were moved to a forced draft turning 
incubator, maintained at the same conditions indicated above, and divided into groups. Six 
experimental groups were used in this study: Ethanol, Saline, GDNF, GDNF+Ethanol, 
BDNF, and BDNF+Ethanol. Embryos received daily injections of ethanol, saline, a NTF, 
or a combination of ethanol and an NTF from ElO to E15. At E16, embryos were removed 
from the eggs, sacrificed by decapitation, and the lumbar section of the spinal cord 
removed and prepared for histology. 

Injections 

Ethanol and saline injections were administered daily from ElO through E15. 
These dates were chosen to replicate a previous study from this laboratory in which ethanol 
was shown to reduce motoneuron number in the lumbar spinal cord (Bradley et al., 1997), 
and because NOCD, which occurs in the chick spinal cord from E5 through E9 (Pittman 
and Oppenheim, 1978), is completed at this time. Since NOCD is completed when 



56 

injections begin, any change in motoneuron number observed is attributable to treatment 
per se and not an interaction of treatment with NOCD. Ethanol embryos received 150 pil of 
30% w/v ethanol (45 mg ethanol per day), dissolved in a 0.9% w/v nonpyrogenic salme 
vehicle, through a pinhole in the shell into the airspace. Previous work in our laboratory 
has detennined that this concentration of ethanol produces blood ethanol counts that peak 
between 250 and 300 mg/dl (Bradley et al., 1997). Salme embryos received 150 pi\ of the 
0.9% w/v nonpyrogenic salme vehicle. NTF injection [GDNF (Amgen) or BDNF 
(Regeneron)], which also occurred from ElO through E15, involved creating a pinhole 
directly over the embryo in addition to the pinhole created in the airspace. The airspace was 
then allowed to shift to a position superior to the embryo and 50 pA of 0.2 mg/ml NTF was 
injected into that space above the embryo ( 10 pig GDNF or BDNF per day). These levels 
of GDNF and BDNF administration were previously shown to rescue motoneurons from 
NOCD without being toxic to the developing embryo (Oppenheim et al., 1995; Oppenheim 
et al, 1992). GDNF+Ethanol and BDNF+Ethanol embryos were given ethanol injections 
from ElO to E15 and NTF injections from ElO to E15 as described above. Ethanol 
mjections preceded NTF injections and the embryos were allowed to sit in a Marsh 
incubator for a period of one hour between injections. This delay in injection time was 
necessary to ensure that the ethanol was absorbed through the inner shell membrane within 
the airspace before the eggs were turned on their side for NTF administration. Also, the 
two injections administered in this study represent a significant volume (200 }A) for the 
embryonic system to incorporate on a daily basis. The delay between injections, therefore, 
allowed absorption of the volume of ethanol before the NTF solution was presented to the 
embryo. Pinholes created by the injection process were sealed with paraffin immediately 
following injection to prevent evaporation and/or leakage of the solutions. The eggs were 
then returned to the turning incubator. 



51 



Dissections and Histological Procedures 

Embryos of all experimental groups were sacrificed by decapitation on El 6 and the 
lumbar section of the spinal cord removed. The vertebrae of the spinal cords were cut 
along the dorsal surface to expose the nervous tissue and allow the fixative to adequately 
penetrate the tissue. Following dissection, the E16 spinal cords were placed in Bouin's 
Fixative for 14 to 21 days to allow the vertebrae to decalcify (Li et al., 1994). The tissue 
was then embedded in paraffin, cut mto 12 pirn coronal sections, mounted onto glass 
slides, and stained with hematoxylm and eosin. 

Motoneuron Size and Spinal Cord Leng th 

Motoneuron size and spinal cord length were measured to determine whether 
ethanol alters any general characteristics of the motoneuronal system. Motoneuron size 
was determined by measuring the diameter of 10 random cells in the same rostral-caudal 
position of the region of each embryonic spinal cord with an eyepiece micrometer. The 
section exactly 2400 /<m following the beginning of the lumbar spinal cord was sampled. 
Three embryos from each experimental condition were analyzed for a total of 30 cells per 
condition. Spinal cord length was determined by counting the number of sections present 
in each embryo following determination of the boundaries of the lumbar spinal cord by the 
anatomical methods described previously and multiplying this number by the section 
thickness (12 /im). 

Motoneuron Counts 

Motoneuron counts were completed following methods described previously 
(Hamburger, 1975; Heaton and Bradley, 1995). Bnefly, a uniform area encompassed by 6 
DRG was noted in each embryo. This procedure ensured that a similar area was counted in 
each subject. Starting from the most rostral section included in the 6 DRG region, 
motoneurons in the lateral motor column of one side of every tenth section were marked 
onto paper using a camera lucida. At 400X magnification, motoneurons were identified in 



58 

the lateral motor column by their large size, dark cytoplasm, and nucleolus. Laterality was 
maintained throughout each individual embryo, but chosen at random before beginnmg the 
countmg process. Previous studies have shown that there is no difference between the 
number of motoneurons contained m the right and left sides of the spinal cord (Pittman and 
Oppenheim, 1979). Each embryo was coded so that the experimenter had no knowledge of 
its experimental treatment until the study was completed. 

Statistical Analyses 

Two-way analysis of variance was performed using SAS version 6. 12 on a 
Pentium computer. When applicable, individual differences between groups were tested 
using Fisher's protected least significant difference post-hoc analyses. Statistical 
significance was determined to be p<0.05. 

Results 
GDNF admmistration did not seem to harm the embryos since survival was 93% in 
the GDNF group and 69% in the GDNF-^Ethanol group. These rates compare favorably 
with control survival rates where 90% of the Saline group and 60% of the Ethanol group 
survived. BDNF administration had a somewhat different effect upon chick survival. The 
BDNF group survived at a rate of 82% and the BDNF+Ethanol group survived at a rate of 
65%. These results indicate that the level of GDNF and BDNF administered in this study 
is not toxic to overall embryonic survival. 

Motoneuron Size and Spinal Cord Length 

As stated in the Methods section above, motoneuron size and spinal cord length 
analyses were performed to determine whether treatments utilized in these studies altered 
the gross morphology of the embryonic spinal cord. Analysis of variance testing indicated 
that embryos from these experimental groups exhibited no differences in motoneuron size 
due to treatment (F=0. 13, df=29, p>0.9). That is, the motoneuron size of ethanol-treated 
animals was unchanged from that of NTF-treated, or control animals. Analysis of variance 



59 



also revealed that overall lumbar spinal cord length was unaltered by treatment (F=0.44, 
df=40, p>0.8). There w/ere no significant differences in the length of the spinal cord 
region counted among the experimental groups. Table 3-1 displays the data from this 
portion of the study. Taken together, these results suggest that the treatments administered 
in this study did not adversely affect the basic anatomy of the spinal cord. 

Table 3-1. Motoneuron Size and Spinal Cord Length. 

Group Cell Size (/^m) Spinal Cord Length (A<m) 



Saline 



Ethanol 



GDNF 



GDNF+Ethanol 



BDNF 



BDNF+Ethanol 



18.6 + 0.525 



18.9 + 0.508 



18.7 + 0.514 



18.4 + 0.502 



18.6 + 0.433 



18.6 + 0.438 



6020+ 69.0 



5966 + 137.9 



5820 + 154.2 



5856 + 148.9 



5904+ 88.2 



5900+ 153.1 



All values are means + SEM. Motoneuron size was detennined by measuring the diameter 
of 10 random cells in the same rostral-caudal position of the region of three embryonic 
spinal cords with an eyepiece micrometer. Therefore, n=30 for each group. Spinal cord 
length was computed by determining the number of sections present in a given spinal cord 
and then multiplying by section thickness (12 }4m). For spinal cord length. Saline n=12 
Ethanol n=7, GDNF n=6, GDNF+Ethanol n=5, BDNF n=5, and BDNF+Ethanol n=6. ' 

Motoneuron Number 

Two way analysis of variance indicated a significant effect due to neurotrophic 
factor administration (F=5.645, df=40, p<0.05) and to ethanol treatment (F=8.902, df=40, 
p<0.005), but not an interaction between neurotrophic factors and ethanol (F= 1.708, 
df=40, p>0.15). Further analysis limiting the groups to Saline, Ethanol, GDNF, and 
GDNF+Ethanol found similar results. Analysis of variance found a significant effect due 
to GDNF treatment (F=9.143, df=29, p<0.01), ethanol treatment (F=6.841, df=29, but 
not an interaction between the two groups (F=0.786, df=29, p>0.35). Limiting the groups 
to Saline, Ethanol, BDNF, BDNF+Ethanol did not produce similar results. Analysis of 
variance found a significant effect due to Ethanol treatment (F=4.381, df=29, p<0.05), but 
not to BDNF treatment (F=0.671, df=29, p>0.4), or an interaction between ethanol and 



60 



BDNF (F=2. 182, df=29, p>0. 15). Figure 3-1 displays the results of these cell counts. 
Post hoc testing revealed significant differences among the groups. Specifically, Ethanol 
embryos contained significantly fewer numbers of motoneurons than GDNF (p<0.0005), 
GDNF+Ethanol (p<0.05), and Saline (p<0.01) treated embryos. There were no significant 
differences among the GDNF, GDNF+Ethanol, or Saline groups. Likewise, there were no 
significant differences among the BDNF, BDNF+Ethanol, Saline, or Ethanol groups. The 
above results suggest that GDNF significantly increased motoneuron number in a manner 
that is independent of ethanol. BDNF did not provide significant protection from ethanol. 
In addition to comparing motoneuron number among the various groups in this study and 
performing a two way analysis of variance for NTF and ethanol treatment, a contrast 
analysis was performed to determine if there was an interaction between GDNF and ethanol 
or between BDNF and ethanol. This analysis found that there was not a significant 
difference between the difference of GDNF from Ethanol and BDNF from Ethanol 
(p>0.5). The conclusion of this analysis is that there was no interaction between GDNF 
and Ethanol or between BDNF and Ethanol. Stated another way, the action of ethanol is 
independent of the action of either GDNF or BDNF. Figure 3-2 illustrates this conclusion. 
The slope of the two lines in the figure are not significantly different This result is 
important because it suggests that the NTFs used in this study are not impaired by the 
actions of ethanol. This analysis is further evidence that ethanol acts independently of the 
NTFs used in the present study. 

Figure 3-3 and Figure 3-4 display photographs of spinal cords taken from the 
animals descnbed in this chapter. Specifically, Figure 3-3 displays photographs of Saline, 
Ethanol, GDNF, GDNF+Ethanol, BDNF, and BDNF+Ethanol spinal cords at40x 
magnification. The overall shape of the spinal cord is altered somewhat by ethanol 
treatment as fewer motoneurons are present and there is less of a "bulge" on the outer edge 
of the cord. Figure 3-4 displays 200x magnification views of the same spinal cord 



61 



2000 



CZ3 

i 



^ 
^ 

^ 



5 

;2; 



1000 




Saline Ethanol GDNF GDNF+ BDNF BDNF+ 

Ethanol Ethanol 

Figure 3-1. Number of motoneurons in the later motor column of the 
lumbar spinal cord at E16. Motoneuron counts are displayed as means 
+ SEM. Data represent actual counts obtained from the lumbar spmal 
cords and are not population estimates. All injections were 
administered from ElO to E15. * = statistical significance in 
comparison to Ethanol; saline (p < 0.01), GDNF (p < 0.0005), and 
GDNF+Ethanol (p < 0.05). Number of animals used for the experiment 
equal to 12 Saline, 7 Ethanol, 6 GDNF, 5 GDNF+Ethanol, 5 BDNF 
and 6 BDNF+Ethanol. 



62 



"3 

0) 



;=5 



O 

CD 

S 

o 
o 



1500 



1300- 



1100 



GDNF+Ethanol 




BDNF+Ethanol 



Ethanol 



_L 



NTF 



NTFand 
Ethanol 



Ethanol 



Interaction Slope 

Figure 3-2. Interaction between ethanol and neurotrophic factors. This 
figure represents the slope of the interaction between GDNF and ethanol 
and between BDNF and ethanol. There was not a significant difference 
between the GDNF group and the BDNF group which suggests that the 
action of each NTF and ethanol is independent. Number of animals 
used for the experiment equal to 12 Saline, 7 Ethanol, 6 GDNF, 5 
GDNF+Ethanol, 5 BDNF, and 6 BDNF+Ethanol. 



Figure 3-3. Photomicrographs of coronal sections from the midlumbar region of E16 
spinal cords. A. Saline, B. Ethanol, C. GDNF, D. GDNF+Ethanol, E. BDNF, and 
F. BDNF+Ethanol. The only noticeable difference is in the density of the and number of 
the motoneurons present in the lateral motor column where the Ethanol group appears to 
have fewer motoneurons present. Number of animals used for the experiment equal to 12 
Saline, 7 Ethanol, 6 GDNF, 5 GDNF+Ethanol, 5 BDNF, and 6 BDNF+Ethanol. 



64 













0, -rii 












Saline 






IT 



\^ 






i:^ 









Ethanol 




my. 




W^.i" ''i^w 



GDNF 



^D 













'^^>^;^''^^?^'''**''^. 



'-i 



.>, 






' >f 









, ^Jn^ ^^' i^ 



GDNF+Ethanol 



■ ■ • .■ ■ ■ ; . ■(■>"'■ 







' fe, -':■■■■ ■^/i'>vC ■■'-■■■ ^v-;^'- V*'?'*'- 



BDNF 




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■■•-•■a :" '^f "■ • . 



BDNF+EthanoI 



... . .■■ V',,'./?-! 




Figure 3-3. 



Figure 3-4. High magnification photomicrographs from the midlumbar section of E16 
spinal cords. A. Saline, B. Ethanol, C. GDNF, D. GDNF+Ethanol, E. BDNF, and 
F. BDNF+Ethanol. This view gives a better perspective of the motor column of each 
animal. The difference in the number and density of the Ethanol animals is apparent. 



66 




Saline 



Ethanol 








'-> 






p 1^;^ 



# 






i^^-' 



1~» f 

' , '.It 



GDNF 



GDNF+Ethanol 





•^^^J;»^MM#rf?? 



*.;*'t5.'' <4,|^ 







BDNF 



BDNF+Ethanol 



Figure 3-4. 



67 

sections. The overall density and number of motoneurons in the lateral motor column again 
appears reduced in comparison to the other groups. 

Discussion 

The major finding of this study is that GDNF can significantly increase motoneuron 
number in the lumbar spinal cord in a manner independent to ethanol (see Figure 3-1). In 
vivo neuroprotection from ethanol neurotoxicity was not demonstrated in embryos treated 
with BDNF. The results of the two way analysis of variance indicated a significant effect 
due to ethanol and NTF treatment. Further analysis of the groups indicated that only 
GDNF administration had a significant effect upon motoneuron number while BDNF did 
not. An interaction between either GDNF and ethanol or BDNF and ethanol was not 
mdicated by this powerful statistical test. Thus, the increase in motoneuron number by 
GDNF is not due to some action of GDNF upon ethanol, but rather GDNF increases 
motoneuron number in a manner that is independent of ethanol toxicity. Ethanol and NTFs 
were administered during a period of development when NOCD was complete, but when 
motoneuron number can be diminished by ethanol exposure. Therefore, NOCD did not 
provide an added variable for this study. These results support previous research from 
other laboratories which found that GDNF could protect certain neuronal populations from 
various neurotoxic events. For example, GDNF protected rat nigral dopamine neurons 
against 6-OHDA lesion in vivo (Choi-Lundberg et al., 1997; Kearns and Gash, 1995; 
Tomac et al., 1995). Also, GDNF prevented death of spinal cord motoneurons following 
axotomy in the chick (Houenou et al., 1996; Oppenheim et al., 1995) and rescued facial 
motoneurons following axotomy in the rat (Gimenez y Ribotta et al., 1997). 

The receptor thought to be responsible for GDNF's activity in the nervous system 
is c-ret and the high affinity receptor for BDNF is trkB. Recent studies suggest that 
GDNF, GDNFRoc, and c-ret form a complex that allows c-ret to transduce the signals from 
GDNF (Jing et al., 1996; Treanor et al., 1996). The ontogeny of the receptors for GDNF 
and BDNF follow different patterns during the development of the neuromuscular system. 



68 

C-ret mRNA expression is detectable, albeit very weakly, in chick spinal cord motoneurons 
as early as E5 and increases throughout development (Nakamura et al., 1996; Schuchardt 
et al., 1995). By E17, c-ret mRNA expression in the chick is expressed at very high levels 
m spinal cord motoneurons (Nakamura et al., 1996; Schuchardt et al., 1995). Therefore, 
c-ret is expressed by the desired target, motoneurons, during the exposure period of the 
present study (ElO to E15). TrkB mRNA is first detectable at E8 and its level of 
expression increases throughout development (McKay et al., 1996). At E16, trkB mRNA 
is highly expressed in the lateral motor column of the spinal cord of the chick (McKay et 
al., 1996). Therefore, trkB is expressed by motoneurons during the exposure period of the 
present study. Since both receptors are expressed during the period of exposure used in 
the present study, another reason must explain the fact that GDNF increases motoneuron 
number from ethanol toxicity whereas BDNF does not. 

In culture chick motoneurons are supported by GDNF (Gouin et al., 1996) whereas 
they are not supported by BDNF (Arakawa et al., 1990). This is not the case in cultures of 
rat motoneurons where BDNF does support their growth (Henderson et al., 1993). Even 
though both GDNF and BDNF prevent NOCD in spinal cord motoneurons, these cells are 
rescued from NOCD to a greater extent by GDNF than BDNF (Oppenheim et al., 1995; 
Oppenheim et al., 1992). This fundamental advantage of GDNF over BDNF to support 
chick motoneurons in culture may explain the results of the present study. The advantage 
of GDNF to support motoneurons to a greater degree than BDNF is supported by the 
results of knockout studies. As was mentioned above, GDNF-deficient, but not BDNF- 
deficient mice exhibit reduced motoneuron number (Jones et al., 1994; Moore et al., 1996). 
Again, the implication of those studies is that GDNF is required to a greater degree for 
proper motoneuron development than is BDNF. The present study found that GDNF 
administration concurrent with developmental ethanol exposure increased motoneuron 
number in a manner independent of ethanol, while BDNF did not significantly alter ethanol 
toxicity. This latter portion of the statement is supported by the fact that two way analysis 



69 

of variance testing did not find a significant effect due to BDNF treatment or an interaction 
between ethanol and BDNF. 

Further experiments using animals genetically altered to overexpress GDNF might 
provide further information about the nature of the increase in motoneuron number 
following embryonic ethanol exposure. By admmistering ethanol to these animals, 
researchers could examme whether GDNF produced by the animal itself could increase 
motoneuron number following ethanol exposure. If these animals proved to be more 
resistant to ethanol insult, it would suggest that some mechanism of the endogenous NTF 
naturally protects developing motoneurons from ethanol to some extent, provided an 
interaction between ethanol and the NTF is demonstrated. Obviously, the normal 
endogenous activity of NTFs in the nervous system does not protect developing chick 
motoneurons from ethanol toxicity since exposure from ElO to E15 reduces motoneuron 
number (Bradley etal., 1997). 

Another line of inquiry could be into cell death genes and the roles they play in NTF 
neuroprotection. Ethanol is known to induce apoptosis in culture (Bhave and Hoffman, 
1997; De et al., 1994; Ewald and Shao, 1993; Liesi, 1997) and in vivo (Rents et al., 1996; 
Singh et al., 1995). The bcl-2 family of cell death molecules has been shown to be 
involved m apoptosis (Boise et al., 1993; Hockenbery et al., 1990). Members of the bcl-2 
family include bcl-2, bcl-Xg, bcl-X^, and bax (Boise et al., 1993; Oltavi et al, 1993). Bcl- 
2 is a membrane-associated protein that interferes with apoptotic cell death. Bcl-Xg acts to 
antagonize bcl-2 activity and promote cell death. Bc1-Xl acts in much the same way as bcl- 
2 and bax binds bcl-2 to inhibit its abihty to prevent apoptosis (Davies, 1995). To 
determine whether the bcl-2 family is involved in GDNF increasing motoneuron number in 
the presence of ethanol, enzyme-linked immunosorbent assay could be used to determine 
precise levels of these molecules in motoneuron cultures exposed to ethanol and GDNF. 
The experiments would be designed to determine whether GDNF added to these cultures in 
the presence of ethanol induces greater expression bcl-2 and bcl-X, -which both prevent 



70 

apoptosis-or decreases expression of bcl-Xg and bax--which both oppose the protective 
activity of bcl-2. The results from these proposed experiments would complement the 
results of the current experiments since they could provide a potential mechanism for 
neuroprotection, should it happen to be demonstrated in the future, by GDNF. It is 
important to note that such a mechanism could proceed independent of ethanol since ethanol 
could conceivably induce apoptosis by another mechanism such as a change in Ca^* 
homeostasis (Koike and Tanaka, 1991; Webb et al, 1995). 

Evidence linking the roles of NTFs and cell death genes has been examined in 
previous studies. Bcl-2 is required for the survival of PC- 12 cells dependent on BDNF but 
not required for survival of CNTF dependent cells (AUsopp et al., 1995). Similarly, bcl-2 
expression is required for the survival of NGF-dependent PC- 12 cells (Katoh et al., 1996). 
Furthermore, NGF has been shown to increase bcl-2 expression in a dose-dependent 
manner in providing this trophic support for these cultured cells (Katoh et al., 1996). A 
similar effect is observed in neuronal cells in that cultured trigeminal ganglion and 
trigeminal mesencephalic neurons are rescued from cell death due to withdrawal of NGF, 
BDNF, or NT-3 by overexpression of bcl-2 (AUsopp et al., 1993). NTFs are related to 
and alter expression of proteins that promote cell death. Withdrawal of trophic support did 
not result in death of axotomized facial motor neurons in bax-deficient mice (Deckwerth et 
al., 1996). The above examples demonstrate the link between NTFs and cell death genes 
of the bcl-2 family. 

The methodology for determining motoneuron number employed m this study has 
been used successfully for determining motoneuron number in this laboratory and others 
(Bradley et al., 1997; Heaton and Bradley, 1995; Oppenheim et al., 1995; Oppenheim et 
al., 1992; Pittman and Oppenheim, 1979). The results indicate that while ethanol 
administration does have an adverse effect on the motoneuron population of the lumbar 
spinal cord, it does not change the overall morphology of the cord. This claim is supported 
by the fact that lumbar spinal cord length, which is an indicator of spinal cord volume, and 



71 

average motoneuron size are unchanged following ethanol treatment. A previous study 
from another laboratory also found that administration of exogenous NTFs from E9 to E15 
did not alter either motoneuron size or spinal cord length measurements (Qin-Wei et al, 
1994). NTFs administered in that study included BDNF, TGF-B, basic fibroblast growth 
factor, and ten other growth factors (Qin-Wei et al., 1994). The present study supports 
this finding and has found that exogenous GDNF or BDNF does not alter these 
relationships since there were no significant differences between any of the exp)erimental 
groups when motoneuron size and spinal cord length were analyzed (See Table 3-1). 

A portion of the current results are somewhat inconsistent with previous research 
(Oppenheim et al, 1995) in that the present study found that motoneuron number in 
GDNF-treated embryos did not differ significantly from control embryos. That study 
found that exogenous GDNF administered from E9 to E15 resulted in significantly more 
motoneurons in the lateral column of the spinal cord than in control embryos (Oppenheim et 
al., 1995). A likely explanation for the differences between these two studies is that 
GDNF injections began on different days. Recall that NOCD continues in the spinal cord 
through E9 (Pittman and Oppenheim, 1979). By beginning GDNF injections on E9, the 
earlier study may have rescued some motoneurons that were destined to undergo NOCD 
and sustained them until E16. The Oppenheim et al. study (1995) found a 12.5% increase 
in motoneuron number, while the present study found an 8%, but not statistically 
significant, increase in motoneuron number in comparison to control embryos. Therefore, 
the absolute difference between the two studies is relatively minimal. 

Previous studies led to the hypothesis that ethanol-induced hypoxia may cause 
excitotoxic damage to developing neurons (Altura et al., 1983; Auer et al., 1989; Barnes 
and Walker, 1981; Bonthius and West, 1990; Jones et al., 1981; Jorgensen and Diemer, 
1982; Michaelis, 1990; Mukherjee and Hodgen, 1982; Pierce et al., 1989). NTFs have 
been shown to prevent hypoxic/ischemic damage in neurons in research performed in other 
laboratories. For example, BDNF has been shown to prevent ischemia-induced cell death 



72 



m rat hippocampal slice cultures (Pringle et al., 1996). Even though previous research has 
not explicitly supported the role of hypoxia in ethanol toxicity, prevention of 
hypoxia/ischemia is another possible mechanism that could explain GDNF's increase of 
motoneuron number in the presence of ethanol. Previous research has not searched for a 
link between ethanol and hypoxia in the developing chick. Hypoxia alone has been studied 
in this animal model. Hypoxic conditions were found to reduce the overall vascularity of 
the chorioallantoic membrane (Strick et al., 1991) and to reduce its blood flow (Ar et al, 
1991). The fact that the direct relationship between ethanol and hypoxia has not been 
explored in the chick does not mean hypoxic conditions do not occur in ethanol -exposed 
chicks. Perhaps a future aim of this research should be to determine whether or not this 
relationship does exist in developing chick embryos. 

As was discussed earlier in the Introduction section of this chapter, the chick 
embryo exhibits many of the chai-acteri sties found in mammalian models of FAS. 
Morphologically, chick embryos treated with ethanol are smaller than controls and have 
decreased brain weights (Pennington and Kalmus, 1987). These similarities between avian 
and mammalian FAS also include molecular changes attributed to ethanol treatment. For 
example, ethanol decreases kinase activities in whole brain of both the chick and rat 
(Kruger et al., 1993; Pennington, 1990) and alters cyclic AMP levels in both chick whole 
brain and in rat striatum (Lucchietal., 1983; Pennington, 1990). Additionally, 
neurotransmitter synthesis is altered by ethanol administration in both the chick and rat 
(Brodie and Vernadakis, 1990; Swanson et al., 1994; Swanson et al., 1995). Other 
molecular mechanisms attnbuted to ethanol in culture are potentially occumng in the avian 
model of FAS. These include those discussed previously such as hypoxia/ischemia (Zajac 
and Abel, 1992) and apoptosis (Rems et al., 1996; Singh et al., 1995). Other mechamsms 
implicated in FAS include hypoglycemia (Fisher et al., 1986) and generation of free 
radicals (Henderson et al., 1995). Such actions could be responsible for the loss of 
motoneurons in the spinal cord observed following ethanol exposure from ElO to E15 



73 

(Bradley et al., 1997). In addition to NTF protection afforded to neurons from apoptosis 
and hypoxia/ischemia, NGF protects cultured rat hippocampal and human cortical neurons 
from hypoglycemic damage (Cheng and Mattson, 1991) and oxidative damage (Mattson 
and Cheng, 1993). The ability of NTFs to inhibit the processes described above could 
provide the mechanism behind the current finding that GDNF increased motoneuron 
number following ethanol exposure. However, the fact that there was not a significant 
interaction between ethanol and GDNF only allows the following conclusion: GDNF 
increases motoneuron number in the lumbar spinal cord in a manner independent to ethanol 
toxicity. 

NTFs have proven to be versatile molecules with the ability to sustain neurons 
when faced with a variety of potentially deadly insults. Now that an increase in 
motoneuron number by GDNF following ethanol exposure has been demonstrated, 
additional investigations will need to be conducted to further examine this action of NTFs 
on developing motoneurons. All of these analyses should be interpreted bearing in mmd 
that the actions of ethanol and the NTFs studied here did not significantly interact. 
Therefore, their actions may be entirely independent. Other NTFs will be tested for their 
ability to protect motoneurons from ethanol toxicity. In addition, combinations of NTFs 
should be tested to determine whether protection is better than when a given NTF is 
administered alone. Specifically, CNTF and NT-3 are good candidate molecules since they 
are known to promote motoneuron survival following axotomy or during the period for 
NOCD (Lo et al., 1995; Yin et al., 1994). Since GDNF increases the number of 
motoneurons independent of ethanol toxicity, other ethanol -sensitive populations might be 
increased in number following administration of GDNF, or other NTFs. Clearly, this 
phenomenon will have to be investigated further to adequately describe the actions of 
various NTFs are protecting neuronal populations or truly increasing neuron number m a 
manner independent of the action of ethanol. 



74 

In addition to testing other NTFs to determine whether they can provide 
neuroprotection from ethanol toxicity, future studies should attempt to build on results 
obtained from the present study to develop potential therapies for FAS. GDNF inserted 
into an adenovirus vector has already proven effective in preventing facial motoneuron 
death following axotomy (Gimenez y Ribotta et al., 1997). Such a delivery system could 
prove effective in getting GDNF to spinal cord motoneurons in mammals to increase 
motoneuron number following ethanol administration. Future research should also focus 
on determining the mechanism by which this increase in motoneuron number is afforded to 
motoneurons by GDNF. By introducing toxic msults such as hypoxia/ischemia and 
hypoglycemia, GDNF's ability to increase motoneuron number in the presence of ethanol 
should be further defined. Unfortunately, more questions remain to be answered about 
GDNF in the future than are answered by the present study. 



CHAPTER 4 

CHARACTERIZATION OF THE NEUROTROPHIN AND NEUROTROPHIN 

RECEPTOR GENE EXPRESSION IN THE HIPPOCAMPUS FOLLOWING CHRONIC 

TREATMENT AND EARLY POSTNATAL ETHANOL TREATMENT IN THE RAT 

Summary 
Fetal alcohol syndrome (FAS) is caused by maternal consumption of ethanol during 
pregnancy and was first described more than two and one-half decades ago. In recent 
years, the thrust of research in this field has been the search for a mechanism of ethanol 
toxicity. Signal transduction and gene expression studies have allowed researchers to learn 
about how ethanol affects neuronal populations at the molecular level (Davis-Cox et al., 
1996; Gandhi and Ross, 1989; MacLennan et al., 1995; Torres and Horowitz, 1996). 
Another area that has garnered attention in this field is neurotrophic factors (NTFs) and 
their ability to affect, and be affected by, ethanol. Previous research has found that ethanol 
can alter expression of specific genes. Examples of genes regulated by ethanol exposure 
include insulin-like growth factor I (IGF-I) and IGF-II in rat brain (Breese et al., 1994; 
Singh et al., 1996). Both of these genes are decreased followmg ethanol exposure. 
However, ethanol exposure increases NMDA receptor gene expression in cultured mouse 
cortical neurons (Hu et al., 1996). The present study attempted to determine the relative 
expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), tyrosine 
receptor kinase B (trkB), and trkC in the hippocampus following ethanol exposure during 
the prenatal or early postnatal period. TrkA was not analyzed because of its extremely low 
level of expression in the developing hippocampus (Martin-Zanca et al., 1990). Nerve 
growth factor (NGF), although attempted, was not assessed because it produced signals 
that were not quantifiable. The results of our analyses indicated that ethanol administration 
during prenatal development in the rat did not change the genetic expression of BDNF, 



75 



76 

NT -3, and trkB as assessed by quantitative Northern blotting. TrkC expression in male 
animals, but not female animals, exposed to ethanol prenatally was reduced. Expression of 
BDNF, NT -3, trkB and trkC was unaffected by postnatal exposure to ethanol during the 
brain growth spurt (BGS). 

Introduction 

Maternal consumption of ethanol exerts many effects upon the developing nervous 
system (Barnes and Walker, 1981; Jones and Smith, 1973; Miller, 1986; Streissguth et al., 
1991 ; West, 1986). FAS continues to be a problem in Western countries and is diagnosed 
in 1-2 out of every 1000 live births in the United States (Abel, 1995). FAS is characterized 
by low birth weight, decreased memory and learning, hyperactivity, facial dysmorphia, and 
lowered IQ (Jones and Smith, 1973; Streissguth et al., 1991). Children bom to heavy 
drinkers experience a higher incidence of FAS with a 4.3% diagnosis rate (Abel, 1995). 
Previous research suggests that the deficits observed in FAS patients are permanent and do 
not lessen with age (Streissguth, 1993). Taken together, these observations led to the 
assertion that maternal consumption of ethanol is the leading known cause of mental 
retardation in the Western Hemisphere (Bonthius and West, 1988). 

Postmortem analysis of human FAS neuropathology has identified central nervous 
system (CNS) abnormalities which include disorders of laminae of the cerebral cortex, 
cerebellar abnormalities, a reduction of dendritic spines on cortical pyramidal cells, changes 
in hippocampal development, and microcephaly (Clarren et al., 1978; Ferrer and Galofre, 
1987). A major problem of examining human subjects is that variables such as nutrition 
and polydrug use are uncontrolled. Animal models have provided controlled exposure to 
ethanol and have been shown to exhibit CNS deficits and behavioral consequences similar 
to those observed in humans (DriscoU et al., 1990). Animal models are particularly useful 
for gaining insight into the effects ethanol exerts on a molecular scale. These models allow 
researchers to answer questions about ethanol consumption that cannot be answered in 
human studies due to ethical and practical reasons (West et al., 1994). 



77 

The rat is the most widely used model in FAS research. However, a caveat of 
using the rat as a model is its gestational period relative to human development. The rat 
gives birth on what is roughly the equivalent of the beginning of the third trimester in 
humans (Goodlett et al., 1993). Important events, such as the bram growth spurt (BGS)- 
where many functional synapses are made in the nervous system—occur in utero during the 
third trimester in humans and postnatally from P4-P10 in rats (West, 1987). Therefore, 
expenments that incorporate ethanol exposure during gestation or during the BGS in rats 
allow researchers to use this as a model of human third trimester ethanol exposure. 
Exposing rat pups to ethanol postnatally produces deficits that demonstrate the importance 
of the BGS and the sensitivity of the CNS to ethanol during this period. Similar to prenatal 
exposure to ethanol in rats, postnatal exposure can produce loss of cerebellar Purkinje cells 
(Bonthius and West, 1990; Bonthius and West, 1991; Goodlett and West, 1992; West, 
1986; West et al., 1990). A problem inherent to any postnatal exposure paradigm is that 
maternal metabolism of ethanol is removed and the subjects are exposed to ethanol in a 
more "adult" manner. Another problem is delivery of ethanol. Suckling rats cannot be 
coerced into readily consuming ethanol because their entire diet consists of mother's milk. 

Two of the methods for delivering ethanol to newborn rats are artificial rearing 
(AR) and inhalation, both of which have advantages and disadvantages. AR consists of 
fitting a neonatal pup with a gastric fistula and tube, maintaining the pups in cups placed in 
a 40°C water bath, and feeding the pup an artificial milk solution via the tube and fistula. 
The AR method provides constant nutrition and produces no damage to the mucous 
membranes of the subject, but the interaction between mother and pup is removed. 
Additionally, AR is a surgical procedure that can be quite stressful for the neonatal rat. The 
stress induced by AR has been found to produce gliosis in rat cortex (Ryabinin et al., 
1995). Recently, the use of intragastric intubation— a less invasive method of neonatal 
ethanol delivery that allows the pups greater maternal access— also resulted in extensive 
gliosis in parietal cortex (Goodlett et al., 1997). The fact remains that gastrostomy control 



78 

rats— pups undergoing AR surgery, but receiving no ethanol-exhibit significant gliosis 
(Ryabinin et al., 1995) and further research should be conducted to detennine whether 
ethanolper se induces gliosis, or whether specific methods of ethanol delivery are 
responsible. The possibility that the AR procedure in and of itself can produce changes in 
brain structure indicates that results obtained using AR could be difficult to interpret. 

Ethanol vapor inhalation involves placing neonatal rats in a sealed chamber that 
contains circulating air and ethanol vapor. Inhaling ethanol vapors has been theorized to 
have the potential to damage the mucous membranes of the lungs which would then 
interfere with oxygen exchange and general metabolism (Ryabinin et al., 1995). However, 
no evidence of lung damage has been observed in rats exposed in this manner (Bauer- 
Moffet and Altman, 1975). The present study utilized the inhalation procedure because of 
the problems associated with AR. Additionally, a previous study from this laboratory 
defined neurotrophic activity in the hippocampus following ethanol inhalation. Therefore, 
proper continuation of that study requires the use of similar methods of analysis and 
ethanol delivery. Other methods for delivering ethanol to rats postnatally include 
concurrent inhalation of mother and pups, direct injection of neonates with ethanol, and 
delivery of ethanol through mother's milk. This latter method is achieved by substituting 
water with a 10% ethanol solution. A problem associated with delivery of ethanol through 
mother's milk is that pups do not receive the same dose of ethanol as the mother due to 
maternal metabolism. 

Mechanisms suggested by previous research may help to explain ethanol's effect on 
the nervous system (reviewed in West et al., 1994). The ability of ethanol to affect DNA 
methylation in the developing embryo has implications for FAS research and the present 
study. Methylation of DNA in eukaryotic cells occurs at the 5' position of cytosine 
residues and converts them into methylcytosine residues. Repressors and enhancers are 
then hindered from binding to DNA. The end result is thought to be a change in gene 
expression (HoUiday, 1987). This change, however, can either increase or decrease the 



79 

expression of a given gene since methylation does not selectively interfere with repressors 
or enhancers. Previous research from the laboratory of Garro found methylation to be 
decreased in fetal DNA following ethanol exposure to the pregnant dam (1991). 
Additionally, ethanol has been shown to interfere with the activity and O^-methyl guanine 
transferase. This enzyme is important in repairing DNA and its unfettered activity is crucial 
for cell survival (Espina et al., 1988). Although these examples have not been shown to be 
caused by changes in DNA methylation, they do indicate that ethanol has the ability to 
regulate genetic expression. For example, ethanol has been shown to decrease expression 
of BDNF mRNA in the hippocampus following chronic exposure to ethanol in adult rats 
(MacLennan et al., 1995) and increase IGF gene expression in whole brain following 
prenatal ethanol exposure (Breese et al., 1994). All of these studies indicate a role for 
ethanol in changing normal cellular biochemistry by affecting genetic expression. 

As was mentioned above, learning and memory deficits are a common characteristic 
of FAS (Abel, 1995; Jones and Smith, 1973; Streissguth et al., 1991). The hippocampus 
is an important structure with regard to memory and learning in humans and animals 
(Bunsey and Eichenbaum, 1996; Cohen and Squire, 1980). Thus it is not surprising to 
find that the hippocampus is sensitive to ethanol and exhibits reduced pyramidal cell 
number following prenatal ethanol exposure (Barnes and Walker, 1981 ; Bonthius and 
West, 1990). Damage to the hippocampus observed in the rat model following ethanol 
exposure may correspond to similar damage in the hippocampus in humans. The present 
study analyzed NTF and NTF receptor gene expression in this region because altered 
neurotrophic activity was implicated in previous studies following both prenatal and 
postnatal exposure to ethanol (Heaton et al., 1995c; Moore et al., 1996). 

The neurotrophin family of NTFs has been shown to play an important role in the 
development of the CNS and peripheral nervous system (PNS) through involvement in 
neuronal differentiation, survival, and maintenance of basic cellular processes. The 
neurotrophin family includes NGF (Levi-Montalcini, 1951), BDNF (Lei brock et al., 



80 

1989), NT-3 (Maisonpierre et al., 1990), NT-4/5 (Berkemeier et al., 1991; Ip et al., 1992), 
and neurotrophin-6 (Gotz et al., 1994). The trk family of receptors has been shown to be 
the high-affinity receptors for the neurotrophins (Martin-Zanca et al., 1990). Trk receptors 
that interact with neurotrophins include trkA (Kaplan etal., 1991; Kaplan et al., 1991), 
trkB (Klein et al., 1990), and trkC (Cordon-Cardo et al., 1991). Specifically, trkA is the 
preferred receptor for NGF, yet to a lesser extent, both BDNF and NT-3 can bind to it. 
TrkB is the preferred receptor for BDNF and NT-4, but can bind NT-3. And trkC is the 
preferred receptor for NT-3. As mentioned above, the neurotrophins are important to 
normal neuronal functioning. They have been shown to regulate a number of peptides- 
including the expression of other neurotrophins-in the rat septohippocampal system (CroU 
et al., 1994). For example, NGF, BDNF, and NT-3 induce ChAT activity (Aldersonet 
al, 1990; Auberger et al, 1987; Gnahn et al, 1983; Nonner et al., 1996); BDNF increases 
NT-3 activity (Lindholm et al, 1994); and BDNF and NT-3 enhance synaptic transmission 
in Shaffer collateral-CAl hippocampal synapses (Kang and Schuman, 1995). This latter 
result is thought to link neurotrophins to long-term potentitation, the mechanism thought to 
be partly responsible for hippocampal induction of memory (Bunsey and Eichenbaum, 
1996). All of these studies indicate that the neurotrophins are important proteins integral to 
normal neuronal functioning in the septohippocampal system. 

Studies of gene deleted "knockout" mice also suggest the importance of 
neurotrophins to proper nervous system development. In totality, the results of these 
experiments have described deficits in the PNS and CNS. However, in most instances the 
CNS remains largely intact in these embryos. Specifically, among neurotrophin and 
neurotrophin receptor knockout mice, the only groups that resulted in statistically 
significant decrease in CNS neurons were trkB deficient animals (Klein et al., 1993). In 
the trkB knockouts spinal cord motoneurons and facial motoneurons were reduced (Klein 
et al, 1993). The effects of the knockouts in the PNS are quite different. All single gene 
knockout studies have found reduced DRG neuron number (Conover et al., 1995; Crowley 



81 

et al., 1994; Klein et al., 1994; Klein et al., 1993; Smeyne et al., 1994). These results 
support earlier studies that found DRGs to be sensitive to many NTFs and to express the 
receptors for multiple neurotrophins (Buchman and Davies, 1993). NGF knockout mice 
do not survive long postnatally (Conover and Yancopoulos, 1997), and there are reduced 
numbers of superior cervical ganglion, trigeminal ganglion, and DRG neurons (Crowley et 
al., 1994). TrkA knockout mice show the same pattern of neuronal loss that the NGF 
knockout mice have and exhibit high mortality (Smeyne et al., 1994). BDNF knockout 
mice die soon after birth and have decreased numbers of trigeminal ganglion, geniculate 
ganglion, nodose-petrosal ganglion, vestibular ganglion, spiral ganglion, and DRG 
neurons (Conover et al., 1995; Conover and Yancopoulos, 1997). NT -4/5 knockout mice 
are similar to BDNF knockouts and have reduced geniculate ganglion, nodose-petrosal 
ganglion, vestibular ganglion, and DRG neurons but do not die early in postnatal life 
(Conover et al., 1995; Conover and Yancopoulos, 1997). TrkB knockouts exhibit high 
mortality and have the CNS differences which were mentioned earlier and reduced numbers 
of trigeminal ganglion, nodose-petrosal ganglion, and DRG neurons (Klein et al., 1993). 
NT-3 knockout mice expire early in postnatal development display fewer superior cervical 
ganglion, trigeminal ganglion, nodose-petrosal ganglion, vestibular ganglion, spiral 
ganglion, and DRG neurons (Conover and Yancopoulos, 1997; Emfors et al., 1994). 
TrkC knockout mice have a high mortality rate, do not survive for a very long period of 
time and exhibit reduced numbers of DRG neurons (Klein et al., 1994). While gene- 
targeting studies are powerful tools for inferring the actions of NTFs and their receptors in 
the nervous system, these studies are not without their difficulties. Changes in the relative 
expression of other genes and changes in the genetic background can have a large effect on 
the development of the knockout animal (Gerlai, 1997). It would be difficult to determine 
whether the observed changes in the organism were due to loss of the gene of interest or to 
a change m genetic background. Therefore, knockout studies must be interpreted with 
these difficulties in mind. 



82 

Important for the present study is the fact that gross neurotrophic responsiveness 
and activity in the septohippocampal system are changed following prenatal exposure to 
ethanol. Cultures of hippocampal neurons derived from rats prenatally exposed to ethanol 
do not respond to basic fibroblast growth factor (bFGF) as well as control cultures (Heaton 
et al., 1995b). That is, these cultures do not extend neurites as the control cultures do in 
response to bFGF. Specifically, bFGF does not promote neurite outgrowth in 
hippocampal cultures derived from ethanol exposed animals to the extent that it does in 
cultures derived from control animals. This result suggests that NTF receptor expression 
may be decreased in response to prenatal ethanol exposure. Following chronic prenatal 
ethanol treatment (CPET) in the rat, neurotrophic activity— which includes both 
neurotrophin and other NTF activity-is increased in extracts made from the hippocampus 
on P21 and cultured on DRG neurons (Heaton et al., 1995c). The increase in neurotrophic 
activity is specific to this region and age of the rat, and suggests an increase in NTF 
expression as a result of prenatal ethanol exposure. No single NTF is implicated by this 
study since DRGs respond to a variety of NTFs in vitro. Postnatal ethanol exposure— in 
contrast to prenatal exposure— produces a reduction in neurotrophic activity of P21 
hippocampal extracts (Moore et al., 1996). Taken together, all of these results suggest a 
role for both the NTF and its receptor in ethanol toxicity. The present study focuses on the 
neurotrophin family of NTFs and their receptors because these proteins are expressed at 
their highest levels in the hippocampus and because they have been implicated as important 
factors for normal septal and hippocampal functioning (Maisonpierre et al., 1990). The 
neurotrophins are not the only NTFs produced by the hippocampus. Other factors that the 
hippocampus is responsive to-such as bFGF (Walicke, 1988)— are synthesized there and 
could affect hippocampal neurons (Ernfors et al., 1990; Riva and Mocchetti, 1991). 

The objective of the present study was to determine whether CPET and early 
postnatal ethanol treatment (EPET) alter the gene expression of neurotrophins in the 
hippocampus of treated rat pups. Thus, this portion of the study relates to the overall 



83 

scheme of this doctoral research by determining how ethanol affects NTF and NTF 
receptor gene expression in vivo. In order to specifically determine whether BDNF, NT-3, 
trkB, and trkC were affected by CPET and EPET, Northern blots were constructed from 
the hippocampi of treated P21 rats. This age was chosen because previous studies found a 
change in gross neurotrophic activity following both prenatal and postnatal ethanol 
exposure that was limited to P21 (Heaton et al, 1995c; Moore et al., 1996). TrkA is 
expressed at very low levels in the hippocampus and was therefore not examined in this 
study (Martin-Zanca et al., 1990). While NGF expression is above the threshold of 
detection for Northern blotting at this age (Maisonpierre et al., 1990), we were unable to 
examine its expression as the resulting bands on our blots were not quantifiable. Repeated 
attempts at probing failed to produce usable data. This age (P21) was chosen for the 
analysis because it coincided with the age of the animals that displayed the alteration of 
neurotrophic activity following prenatal and postnatal ethanol exposure (Heaton et al., 
1995c; Moore et al., 1996). Relative expression of these genes was compared between 
control and ethanol -exposed animals. Following CPET, male animals exhibited reduced 
gene expression of trkC while female animals exhibited no significant differences. There 
were also no significant differences in gene expression in female CPET animals or 
following EPET. 

Materials and Methods 

Prenatal Ethanol Exposure 

Long-Evans hooded rats originally obtained from Charles River Company were 
used to establish a breeding colony. Animals were housed individually in plastic cages 
under controlled temperature and humidity conditions. NuUiparous females were placed in 
a cage with an experienced male. On the following morning pregnancy was determined by 
the presence of sperm following vaginal lavage. Animals were placed on one of three diets: 
Chow, Ethanol, or Sucrose (n=24 for each group). The Chow group was given access to 
Purina Rat Chow and water ad libitum. The Ethanol group was given free access to an 



84 

ethanol-containing liquid diet in which ethanol comprised 36% of the total caloric intake 
(ethanol concentration = 8.4% v/v). The Sucrose group was pair-fed the same volume of 
liquid diet with an isocaloric substitution of sucrose for ethanol. The liquid diet was made 
from a commercial formula, Sustacal (Mead Johnson), which was supplemented with 
Vitamin Diet Fortification Mixture (3.0 g/liter) and Salt Mixture (5.0 g/liter; both from ICN 
Nutritional Biochemicals). The liquid diets contained 1.3 kcal/ml and provided several 
times the daily requirements of all essential vitamins and nutrients. The additional 
fortification ensured proper nutritional intake, so that any results obtained from the ethanol- 
treated animals could be directly attributed to ethcinol per se, and not to possible nutritional 
deficiencies. Ethanol and Sucrose pups were fostered to Chow dams on the day of birth to 
remove any possible effect the diet might have on the ability of the dam to properly rear the 
pups. Chow pups were left with their birth mother. Previous experiments in this 
laboratory have shown that morning blood alcohol levels of the pregnant dams range from 
1 12 mg/dl to 254 mg/dl (Swanson et al., 1995). 

Postnatal Ethanol Exposure by Inhalation 

Pregnant Long-Evans hooded rats obtained from Charles River Company were 
used for this portion of the study. Dams were fed standard lab chow throughout the 
experiment, ad libitum. Pups from these litters were placed into one of three groups: 
Ethanol, control Separated, and control Unseparated (n=24 for each group; these groups 
will be referred to as Ethanol, Separated, and Unseparated, respectively, for the remainder 
of this chapter). Ethanol pups never came from a litter that contained another group; 
however, Separated and Unseparated litters were split so that one dam nursed equal 
numbers of pups from each group. Ethanol litters were culled to 7 pups on postnatal day 
(P4), while Separated/Unseparated litters were culled to 10 pups (Ryabinin et al., 1995). 
The reduced number of pups in the Ethanol group was done to help eliminate nutritional 
differences between ethanol -exposed and control pups. Ethanol inhalation occurred from 
P4 through PIO. Pups were placed in the inhalation chamber on a heating pad at 37°C and 



85 

were allowed to breathe ethanol vapor in a chamber for 2 hours daily. The inhalation 
chamber consisted of an airtight 10-gallon aquarium fitted with an intake and out-take hose. 
The intake hose received air flowed into a 1 L Erlenmeyer vacuum flask containing 520 ml 
95% ethanol (Aaper) from an aquarium air pump set to pump air at approximately 0.8-1 
L/min. Ethanol used in this study, and all studies in this project, was not treated with 
benzene or any other chemical known to exert detrimental effects upon the nervous system. 
As the air was forced into the flask it passed through a 1.5 inch air stone submerged in the 
ethanol. The ethanol-laden vapor was then carried to the chamber. The out-take hose led 
ethanol vapor from the chamber to a fume hood. Separated pups were placed for 2 hours 
daily in a similar chamber with the difference being that air was pumped directly into the 
chamber from the air pump without encountering ethanol. Unseparated animals remained 
with the nursing dam while the Ethanol and Separated pups were placed in their respective 
chambers. This paradigm of ethanol exposure resulted in peak blood ethanol counts of 
approximately 250 mg/dl. Ethanol pups were clearly intoxicated upon removal from the 
chamber and remained incapacitated, and unable to nurse, for a period of approximately 
two hours following exposure. In contrast, Separated pups began nursing immediately 
upon their return to the home cage. 

Morphometric Measurements 

Prior to sacrifice, all animals were weighed and had crown-rump length 
measurements taken. Crown-rump length was defined as the distance from the crown of 
the skull— defined to be the point directly between the ears— to the base of the tail. The brain 
of each animal was weighed before the hippocampus was dissected. These measures were 
taken to provide an estimation of the overall effect that ethanol treatment had on the 
subjects. 

Dissections 

Rats were anesthetized with methoxyflurane (Pittman-Moore) and sacrificed by 
decapitation on P21. After the brain was removed from the skull, the hippocampus was 



86 

dissected out, wrapped in aluminum foil, and flash-frozen in liquid nitrogen. Endogenous 
ribonucleases present in the tissue rapidly destroy mRNA present in the brain once a 
dissection starts. Therefore, mRNA (which happens to be the molecule of interest in the 
present study) is in danger of being lost if the dissection is not completed with considerable 
speed. Because of the rapidity with which dissected tissue had to processed and because of 
the considerable time required to obtain tissue weights, individual weights of the 
hippocampi were not taken. The tissue was then stored at -70°C until RNA was extracted. 

RNA Extraction 

Polyadenylated (poly- A) messenger RNA (mRNA) was extracted from frozen 
tissue specimens via the Micro-fastTrack kit (Invitrogen). Poly-A mRNA was stored as a 
precipitate in 75% ethanol at -70°C until the samples were run on an electrophoresis gel. 

Northern Blots 

The procedures used in this study are as previously described (Back et al., 1994; 
MacLennan et al., 1994; MacLennan et al., 1995). The amount of mRNA in each sample 
was assessed by taking the optical density of 1 pil of sample in 500 pil dH^O at 260 nm and 
280 nm of UV light. The samples were then loaded onto a 1.25% agarose formaldehyde 
denaturing gel so that each lane contained about 15 ]Ag of poly-A mRNA. To obtain 
mRNA levels of this magnitude, four animals were used for each lane in each gel. The 
products were separated using horizontal gel electrophoresis rurming at 100 V for 30 
minutes and then turned down to 25 V and allowed to run overnight. The poly-A mRNA 
was then transferred to a nylon membrane (ICN). The membrane was then baked in a 
vacuum oven at 80° C for two hours and stored desiccated at room temperature until probed. 
Northern blots were probed with a cDNA strand encoding one of two neurotrophins (NT-3 
or BDNF), one of two neurotrophin receptors (trkB or trkC), or cyclophilin. Cyclophilin 
mRNA is constituitively expressed and was used to standardize each lane. Previous 
research has shown that cyclophilin gene expression is not affected by developmental 
ethanol treatment (Maier et al., 1996). The cDNA strands were labeled with ^^P (dCTP 



87 

from Amersham) by random hexamer priming. The BDNF, NT-3, and trkB cDNA probes 
were a generous gift from Drs. P. Isackson, J.G. Sutcliffe, and S. Whittemore to Drs. Don 
Walker and A. John MacLennan of this department. The trkC cDNA probe was a generous 
gift from Dr. Louis Parada to this laboratory. Before starting the entire hybridization 
procedure, the blots were prewashed for 60 minutes at room temperature in 2X SSC. The 
blots were prehybndized at 42°C for approximately 24 hours m a solution containmg 50% 
formamide, 5X SSC, 5X Denhardt's, 0.5% SDS, 0.05M sodium phosphate, 0.25 mg/ml 
salmon sperm DNA, and 0. 1 mg/ml poly-A. Hybndization was carried out at 42°C for 
approximately 20 hours in the same solution described above with the ^^P labeled cDNAs 
added to the solution. Following hybridization the blots were washed three times in 2X 
SSC at room temperature for periods of one minute, 30 minutes, and 30 minutes, 
respectively. The blots were then washed twice at 58°C in a solution containing 0. IX SSC 
and 0.5% SDS for 30 minutes. After this final wash the Northerns were wrapped in plastic 
wrap and lightly taped to a Molecular Dynamics phosphorimaging cassette for at least 24 
hours and analyzed by using the ImageQuant program which computes the density of each 
band electronically. The exposure time was dependent on the relative expression of the 
gene being probed. The phosphorimaging cassette is lOX more sensitive to radioactive 
particles than the x-ray film, but records ambient radiation. The ImageQuant program 
allows background readings to be subtracted so that radiation from the probe itself can be 
analyzed. The resulting bands were normalized by dividing each value by the 
corresponding cyclophilin value. 

Stripping and Reprobing 

In order to probe the blots for different neurotrophic agents and remove any 
remaining ^^P from previous hybridizations, the blots were stripped. The blots were 
exposed to a solution containing 50% formamide and O.OIM sodium phosphate at 65°C for 
60 minutes. After stripping, the blots were rinsed in a solution contaming 2X SSC and 



88 

0. 1% SDS for five minutes at room temperature. The blots were then probed as described 
in the previous section. 

Statistical Analyses 

Two-way analysis of variance was performed using SAS version 6. 12 on a 
Pentium computer. Variances were pooled for this analysis since testing revealed that they 
were not significantly different by gender. When applicable, individual differences 
between groups were tested using Fisher's protected least significant difference (PLSD) 
post-hoc analyses. Statistical significance was determined to be p<0.05. Additionally, the 
Bonferroni/Dunn correction was used to determine if individual differences elucidated by 
Fisher's PLSD were valid. Statistical significance following the Bonferroni/Dunn 
correction was p<0.137. 

Results 

Morphometric Measurements 

Morphometric measurements were obtained to assess the general effect that ethanol 
had on the development of the animals used in this study. Measurements of body weight, 
brain weight, and crown-rump length were collected from both prenatally and postnatally 
treated animals on P21. The postnatally exposed rats were weighed on a daily basis from 
P4 to PIO to assess their overall growth during the inhalation period. Each group in both 
the CPET and EPET studies contained 24 animals and sexes were kept separate during all 
analyses. 
CPET 

As mentioned above, measurements were taken from both male and female animals 
on P21 . For male animals, analysis of variance found no significant effect due to treatment 
for body weight, brain weight, or crown-rump length. Female animals had a differing 
result. Analysis of variance did not find a significant effect due to treatment for body 
weight or crown-rump length. However, brain weight was significantly affected by 



89 

treatment (F=6.37, df=71, p<0.005). Post hoc testing reveled that Alcohol animals had 
significantly smaller brains than both Chow (p<0.005) and Sucrose (pxO.Ol) animals. 
Even though the brains were significantly smaller in female animals following CPET, the 
ratio of brain weight to body weight was not significantly different from the same ratio in 
control animals. This ratio was also not significantly changed in male animals exposed to 
ethanol prenatally. Figure 4-1 displays the brain weights obtained from this portion of the 
study. Since brain weight to body weight ratio was unaffected by CPET, the extent to 
which brain weight was affected by ethanol exposure is not clear. 
EPET 

Analysis of variance found significant effects due to treatment in both male and 
female animals in this section of the study. In the interest of clarity, these results will be 
presented separately. 

Male animals. Male animals displayed a significant effect of treatment for weight at 
P4 (F=7.05, df=71, p<0.005) and P5 (F=4.06, df=71, p<0.05). At P4, post hoc testmg 
revealed that Ethanol animals weighed significantly more than both Separated (p<0.05) and 
Unseparated (p<0.0005) animals. At P5, Ethanol animals were only significantly larger 
than Unseparated animals (p<0.01). It should be noted that it was not the intent of the 
researchers to select larger animals in one group over the other. A possible explanation for 
this difference may lie in the litter size of the relative groups. Since Ethanol groups were 
set to have 7 pups at the start of inhalation and the control groups (Separated and 
Unseparated) were combined in one litter, litters containing less than 10 pups were 
automatically put into the Ethanol group. When both litters contained more than 10 pups, 
the dam was randomly placed into either a control or Ethanol group. Therefore, the 
Ethanol groups most likely started, on average, with a smaller litter size. This would make 
each pup larger on average than pups bom to larger litters. 

Since there was a difference at the start of treatment, gross initial differences 
between the groups were eliminated by analyzing weight gain from day to day. Analysis of 



90 



A 



B 



DC 

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00 






DJCl 

■ I-H 




Alcohol Sucrose Chow 
CONDITION 
Figure 4-1. Brain weight at P21 of female and male animals following 
prenatal ethanol exposure. A. Female brain weight. B. Male brain weight. 
Measurements are means + SEM. a = Female Ethanol animals are 
significantly smaller in comparison to Sucrose (p<0.01) and Chow (p<0.005). 
Animals were fed Ethanol, Sucrose, and Chow diets during gestation as 
described in the Methods section of this chapter and fostered to chow dams at 
birth, n = 24 for each group. 



91 

variance found a significant effect due to treatment for weight gain in male animals from P5 
to P6 (F=7.85, df=71, p<0.001), from P8 to P9 (F=4.56, df=71, p<0.05), from P4 to 
PIO (F=13.53, df=71, p<0.0001), and from PIO to P21 (F=7.18, df=70, p<0.005). Post 
hoc testmg revealed that the weight gain from P5 to P6 was significantly larger in Sepai^ated 
(p<0.005) and Unseparated (p<0.001) animals than in Ethanol animals. Further, from P8 
to P9, Unseparated animals gained significantly more weight than Ethanol animals 
(p<0.005). Over the entire course of the ethanol inhalation period (from P4 to PIO), there 
were significant differences. Separated (p<0.05) and Unseparated (0.0001) animals gained 
significantly more weight from P4 to PIO than Ethanol animals. Additionally, Unseparated 
animals gained significantly more weight than Separated animals (p<0.01). Figure 4-2 
displays the results observed in this section of the study. Following the inhalation period, 
the Ethanol animals exhibited a growth spurt which effectively "caught them up," and 
allowed them to surpass the other groups. Recall that the number of animals in each litter 
was controlled so that the Ethanol group had 7 pups and the Control group had 10 pups. 
This was done to give the Ethanol group greater food availability and to control for possible 
nutritional differences. Specifically, Ethanol animals gained significantly more weight 
from PIO to P21 than both the Separated (p<0.001) and Unseparated (p<0.005) animals. 
Figure 4-2 also displays these results. 

For male animcds at P21, analysis of variance found that body weight (F=4.7l, 
df=70, p<0.05), crown-rump length (F=4.36, df=70, p<0.05), and brain weight 
(F=21.39, df=71, p<0.0001) were all significantly affected by treatment. For body 
weight, post hoc testing revealed that Ethanol animals weighed significantly more than both 
Separated (p<0.005) and Unseparated (p<0.05) animals. Similarly, Ethanol animals had a 
significantly longer crown-rump length than their Separated (p<0.005) counterparts. These 
results are not surprising in light of the fact that Ethanol animals gained significantly more 
weight from PIO to P21 than both Separated and Unseparated animals. Both Separated 
and Unseparated animals had significantly larger brains than Ethanol animals (p<0.0001 



92 



45- 


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Figure 4-2. Weight gain during postnatal ethanol exposure in male animals. 
Ethanol exposure extended from P4 through PIO. Additionally, body weights at 
P21 are displayed. Measurements are means + SEM. a = Ethanol animals show 
significantly lower weight gain from P5 to P6 in comparison to Separated 
(p<0.005) and Unseparated (p<0.001) animals, b = Ethanol animals display 
significantly lower weight gain from P8 to P9 in comparison to Unseparated 
(p<0.005) animals, c = Ethanol animals exhibit significantly lower weight gain 
from P4 to PIO in comparison to Separated (p<0.05) and Unseparated (p<0.0001) 
animals. Also, Separated animals gained significantly less weight than 
Unseparated (p<0.01) animals, d = Ethanol animals gain significantly more 
weight from PIO to P21 than Separated (P<0.001) and Unseparated (p<0.005) 
animals. Ethanol, Separated, and Unseparated groups are described in the 
Methods section of this chapter, n = 24 for each group. 



93 

for both comparisons). Unlike prenatally exposed animals, this difference in brain weight 
extended to the brain weight to body weight ratio. For this ratio, analysis of variance 
found a significant effect due to treatment (F=13.989, df=:71, p<0.0001) and post hoc 
testing showed that both Separated (p<0.0001) and Unseparated (p<0.0005) had 
significantly larger ratios than Ethanol animals. Figures 4-3 and 4-4 display the results of 
this portion of the study. 

Female animals. Following EPET, female animals exhibited some results which 
were similar to male animals and some that were not. Specifically, analysis of variance 
found that during the period of ethanol exposure, there was a significant effect due to 
treatment on P4 (F=8.74, df=70, p<0.0005) and P5 (F=6.52, df=70, p<0.005). No other 
days during the exposure period showed any significant differences. At P4, Ethanol 
animals were significantly larger than both Separated (p<0.001) and Unseparated 
(p<0.001) animals. At P5, Ethanol animals were again larger than both Separated 
(p<0.01) and Unseparated (p<0.005) animals. During the exposure period, female animals 
did not exhibit as much variability as the male animals did and on only one day did the 
female animals exhibit any effect due to treatment. Analysis of variance found that from P5 
to P6 (F=6.52, df=70, p<0.005), the Separated (p<0.0005) and Unseparated (p<0.005) 
animals gained more weight than the Ethanol animals. Over the course of the entire 
inhalation period (P4-P10), there was also an effect of treatment (F=5.50, df=70, p<0.01). 
Specifically, the Separated (p<0.05) and Unseparated (p<0.005) animals gained more 
weight over the period than the Ethanol animals. After the period of inhalation was 
completed until sacrifice (PIO to P21), analysis of variance approached significance 
(F=2.96, df=69, p=0.059). Figure 4-5 shows the results found in this section of the 
study. 

At P21, when the female animals were sacrificed and prepared for dissection, the 
only measurement that was affected by treatment was brain weight (F=3 1. 13, df=70, 
p<0.0001). Neither body weight nor crown-rump length was affected. Post hoc analysis 



94 



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Condition 

Figure 4-3. Gross morphological measurements following EPET in male 
animals at P21. A. Body weight. B. Crown rump length. Measurements are 
means + SEM. Ethanol, Separated, and Unseparated groups are as described 
in the Methods section of this chapter, a = significantly larger than Separated 
(p<0.005) and Unseparated (p<0.05) animals, b = significantly longer in 
comparison to Separated (p<0.005) animals, n = 24 for each group. 



95 




Ethanol Separated Unseparated 

Figure 4-4. Brain weight and Brain weight to body weight ratio of EPET male 
animals at P21. A. Brain weight. B. Brain weight to body weight ratio. 
Measurements are means + SEM. Ethanol, Separated, and Unseparated groups 
are as described in the Methods section of this Chapter, a = significantly smaller 
than both Separated (p<0.0001) and Unseparated (p<0.0001) animals, b = 
significantly smaller ratio than Separated (p<0.0001) and Unseparated 
(p<0.0005) animals. n= 24 for each group. 



96 



45- 



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15' 



-Q — Ethanol 
-» — Separated 
■^ — Unseparated 




T" 

5 



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4 



10 



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Age 



Figure 4-5. Weight gain during postnatal ethanol exposure in female animals. 
The period of ethanol exposure extended from P4 through PIO. Additionally, 
body weight at P21 is displayed. Measurements are means + SEM. Ethanol, 
Separated, and Unseparated groups are as described in the Methods section of this 
chapter, a = Ethanol animals exhibit significantly less weight gain from P5 to P6 
than Separated (p<0.0005) and Unseparated (p<0.005) animals, b = Ethanol 
animals display significantly less weight gain than Separated (p<0.05) and 
Unseparated (p<0.005) animals, n = 24 for each group. 



97 

found that Separated (p<0.0001) and Unseparated (p<0.0001) animals had larger brains 
than ethanol animals. Brain weight to body weight ratio was affected similarly. Overall, 
there was an effect of treatment (F=14.635, df=68, p<0.0001) and both the Separated 
(p<0.0005) and Unseparated (p<0.0001) animals had a larger ratio than the Ethanol 
animals. Figure 4-6 displays the results obtained in this section of the study. 

Gene Expression of Neurotrophins and Neurotrophin Receptors 

Analysis of gene expression in the hippocampus of rats exposed to ethanol 
prenatally (CPET) and postnatally (EPET) is the major experiment of this chapter. As was 
evidenced by the above data, ethanol treatment did have some effect on the overall growth 
of animals, both prenatally and postnatally. This effect did not necessarily translate into a 
change in gene expression of neurotrophins or neurotrophin receptors. Briefly, while there 
were significant differences in gene expression in male animals following CPET, there 
were none found in female animals. There were no significant differences following EPET 
in either male or female animals. The specific differences found are outlined below. 
CPET 

Analysis of variance found that there were no effects due to treatment in female 
animals when BDNF, NT -3, trkB, and trkC gene expression was analyzed. Male animals 
displayed no significant effect due to treatment when BDNF, NT -3, and trkB gene 
expression was analyzed. Figures 4-7, 4-8, and 4-9 display the results of BDNF gene 
expression for male and female animals. Figure 4-7 displays the phosphorimaging view of 
these blots and Figures 4-8 and 4-9 are bar graphs showing the quantitative data from these 
blots. Figures 4-10 and 4-11 exhibit the results of NT-3 gene expression for male and 
female animals. Figure 4-10 is the phosphorimaging view of the NT-3 Northern blots and 
Figure 4- 11 is a bar graph of NT-3 relative gene expression. Figures 4- 12 and 4- 13 are bar 
graphs depicting the gene expression of trkB active and truncated receptors, respectively. 
Figures 4-14, 4-15, 4-16, and 4-17 exhibit the results of trkC gene expression for male and 
female animals. Figure 4-14 is the phosphorimaging view of the trkC blots and Figures 4- 



98 



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03 

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0.03- 



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Ethanol Separated Unseparated 
Figure 4-6. Brain weight and brain weight to body weight ratio in EPET female 
animals at P21. A. Brain weight. B. Brain weight to body weight ratio. 
Measurements are means + SEM. Ethanol, Separated, and Unseparated groups 
are as described previously in the Methods section of this chapter, a = 
significantly smaller in comparison to Separated (p<0.0001) and Unseparated 
(p<0.0001) animals, b = significantly smaller ratio in comparison to Separated 
(p<0.0005) and Unseparated (p<0.0001) animals, n = 24 for each group. 



Figure 4-7. Phosphorimaging view of BDNF Northern blots composed of the 
hippocampal region from P21 rat brains exposed to ethanol prenatally. Images are obtamed 
from Northern blots composed of male and female animals. Each sex is broken down 
further into Chow, Sucrose, and Ethanol groups as described previously in the methods 
section of this chapter. Two transcripts for BDNF were detected. The larger transcript is 
known to be 4.4 kb and the smaller one is known to be 1.7 kb in length. These sizes were 
confirmed by comparing the transcripts to the darkest band of the ladder. Number of lanes 
used in the analysis equal to 6 for each group. 



100 



Male Female 

Chow Sucrose Ethanol Chow Sucrose Ethanol Ladder 

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1 


L 





P^ 


>-. 


iS 


u 



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a 


a 




CJ 











Ethanol Sucrose Chow 



Figure 4-8. Relative BDNF 4.4 kb transcript expression in rat hippocampus at 
P21 following prenatal exposure to ethanol. A. Female animals. B. Male 
animals. Ethanol, Sucrose, and Chow groups are as described previously in 
this chapter. Measurements are means + SEM. Two way analysis of variance 
revealed no significant differences among the groups. n=6 for each group. 



102 



0.03- 




Ethanol Sucrose Chow 



Figure 4-9. Relative BDNF 1.7 kb transcript gene expression following prenatal 
exposure in P21 rats. A. Female animals. B. Male animals. Ethanol, Sucrose, 
and Chow animals are as described in the Methods section of this chapter. 
Measurements are means + SEM. Analysis of variance indicated no significant 
differences among the groups. n=6 for each group. 



Figure 4-10. Phosphorimaging view of NT -3 Northern blots composed of the 
hippocampal region from P21 rat brains exposed to ethanol prenataUy. Images are obtained 
from Northern blots composed of male and female animals. Each sex is broken down 
further into Chow, Sucrose, and Ethanol groups as described previously in the methods 
section of this chapter. The NT-3 transcript is known to be 1.5 kb in length. This size was 
confirmed by comparing the distance relative to the darkest band of the ladder. Number of 
lanes used in the analysis equal to 6 for each group. 



104 



Male 

Chow Sucrose tthanol 



« 



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ki- 






V^ 



■M^^ 



Female 

Chow Sucrose Ethanol Ladder 



■I 




W^!^^ 



^. 



^?M 




Ic:: 



1.5 kb 









Figure 4-10. 



105 




Ethanol Sucrose Chow 

Figure 4-11. Relative NT -3 gene expression following prenatal ethanol exposure 
inP21rats. A. Female animals. B. Male animals. Ethanol, Sucrose, and Chow 
groups are as described in the Methods section of this chapter. Measurements are 
means + SEM. Analysis of variance indicated no significant differences among 
the groups. n=6 for each group. 



106 



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55 O 

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0.04- 



0.03- 



0.02- 



0.01- 



O.OO 





Ethanol Sucrose Chow 

Figure 4-12. Relative trkB active receptor gene expression following prenatal 
ethanol exposure in P21 rats. A. Female rats. B. Male rats. Ethanol, Sucrose, 
and Chow groups are as described in the Methods section of this chapter. 
Measurements are means + SEM. Analysis of variance indicated no significant 
differences among the groups. n=6 for each group. 



107 



A 0.08- 



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Ethanol Sucrose Chow 



Figure 4-13. Relative trkB truncated transcnpt gene expression following 
prenatal exposure in P21 rats. A. Female animals. B. Male animals. Ethanol, 
Sucrose, and Chow animals are as described earlier in the Methods section of this 
chapter. Measurements are means + SEM. Analysis of variance indicated no 
significant differences among the groups. n=6 for each group. 



Figure 4-14. Phosphorimaging view of trkC Northern blots composed of the hippocampal 
region from P21 rat brains exposed to ethanol prenatally. The largest band of the ladder is 
1.7 kb in length. Images are obtained from Northern blots composed of male and female 
animals. Each sex is broken down further into Chow, Sucrose, and Ethanol groups as 
described previously in the methods section of this chapter. The largest band corresponds 
to the active form of trkC ( 14 kb). The two smaller bands are truncated versions of trkC 
(4.7 kb and 3.9 kb). Size was confirmed by comparing the bands relative to the darkest 
band on the ladder, a = artifact, not a band. Number of lanes used in the analysis equal to 
6 for each group. 



109 



Male 

Chow Sucrose Ethanol 






Female 

Chow Sucrose Ethanol Ladder 



. /%: 




jit 


..-.* 


14 kb 


:''^!',r ?,:';:- 






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no 



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Ethanol Sucrose Chow 
Figure 4-15. Relative trkC 14 kb transcript gene expression at P21 in rats 
exposed to ethanol prenatally. A. Female animals B. Male animals. 
Measurements are means + SEM. Ethanol, Separated, and Unseparated groups 
are as described in the Methods section of this chapter, a = significantly lower 
expression in comparison to Sucrose (p<0.01) and Chow (p<0.005) animals, n : 
6 for each group. 



Ill 



0.06 




Ethanol Sucrose Chow 



Figure 4-16. Relative trkC 4.7 kb truncated transcript gene expression following 
prenatal exposure in P21 rats. A. Female animals. B. Male animals. Ethanol, 
Sucrose, and Chow animals are as described earlier in the Methods section of this 
chapter. Measurements are means + SEM. Analysis of variance indicated no 
significant differences among the groups. n=:6 for each group. 



112 



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Ethanol Sucrose Chow 



Figure 4-17. Relative trkC 3.9 kb transcript gene expression following prenatal 
exposure in P21 rats. A. Female animals. B. Male animals. Ethanol, Sucrose, 
and Chow animals are as described earlier in the Methods section of this chapter. 
Measurements are means + SEM. Analysis of variance indicated no significant 
differences among the groups. n=6 for each group. 



113 

15, 4-16, and 4- 17 are bar graphs for the relative gene expression of the active receptor, 
and two truncated receptors, respectively. The phosphorimaging view of the standard, 
cyclophilin is displayed in Figure 4-18. Previous research has found that there are three 
poly-A transcripts of trkC (Jaber et al., 1994). The probe used to detect trkC in this study 
detected all three of these transcripts. The largest transcript corresponded to the active form 
of trkC and was approximately 14 kb in length. The two smaller transcripts corresponded 
to truncated versions of the trkC receptor and were approximately 3.9 and 4.8 kb in length. 
These sizes are confirmed by comparing the size of the trkC bands to those of BDNF and 
NT -3. The same probes used in an earlier study were used in the present study 
(MacLennan et al., 1995). The BDNF probe detects two BDNF transcripts that are 4.4 kb 
and 1.7 kb in length (MacLennan et al., 1995). The NT -3 probe detects an NT -3 transcript 
that is 1.5 kb in length. The ladder present in each Northern blot is pictured in each 
Northern blot Figure and comparison to the 4.4 BDNF transcript confirms that the two 
smaller trkC truncated transcripts are about 3.9 and 4.7 kb in length. The largest trkC 
transcnpt (14 kb) is cleariy larger than the largest band in the RNA ladder which is 
approximately 9.5 kb in length. In male animals, there was a significant effect due to 
treatment in active trkC gene expression (F=7.0, df=17, p<0.01). Post hoc analyses found 
that the active trkC transcript was decreased in Ethanol animals in comparison to Chow 
(p<0.005) and Sucrose (p<0.01) animals. These comparisons are also significant 
following the Bonferroni/Dunn correction since p<0.0137. These reductions were 
approximately 20% in both instances. 

In addition to analyzing the absolute gene expression, an analysis of the ratios of 
the three trkC transcripts was performed. Analysis of variance found that the ratio of the 
active trkC transcript to the smaller truncated transcript was affected by treatment (F=4.72, 
df=17, p<0.05). Post hoc testing revealed that in both Chow (p<0.05) and Sucrose 
(p<0.05) animals, this ratio was 20% larger than m Ethanol animals. However, the 
Bonferroni/Dunn correction found this latter difference to not be significant. The difference 



Figure 4-18. Phosphorimaging view of cyclophilin Northern blots composed of the \ 

hippocampal region from P21 rat brains exposed to ethanol prenatally. Images are obtained j 

from Northern blots composed of male and female animals. Each sex is broken down j 

further into Chow, Sucrose, and Ethanol groups as described previously in the methods i 

section of this chapter. The transcript is approximately 1 .0 kb in size relative to the darkest i 

band. Number of lanes used in the analysis equal to 6 for each group. ! 



115 



Male 

Chow Sucrose Ethanol 



Female 

Chow Sucrose Ethanol Ladder 





. ' c • ■ 











Figure 4-18. 



116 

m the ratio of active trkC to truncated trkC is probably due to the fact that absolute 
expression of active trkC was significantly smaller in the Ethanol group. 
EPET 

As mentioned above, there were no significant differences in the expression of any 
of the genes analyzed following EPET. Additionally, the ratio of expression of various 
transcripts was unchanged. Figures 4-19, 4-20, and 4-21 display the results of BDNF 
gene expression for male and female animals. Figure 4-19 displays the phosphorimaging 
view of these blots and Figures 4-20 and 4-21 are bar graphs showing the quantitative data 
from these blots. Figures 4-22 and 4-23 exhibit the results of NT-3 gene expression for 
male and female animals. Figure 4-22 is the phosphorimaging view of the NT-3 Northern 
blots and Figure 4-23 is a bar graph of NT-3 relative gene expression. Figures 4-24 and 4- 
25 are bar graphs depicting the gene expression of trkB active and truncated receptors, 
respectively. Figures 4-26, 4-27, 4-28, and 4-29 exhibit the results of trkC gene 
expression for male and female animals. Figure 4-26 is the phosphorimaging view of the 
blots probed for trkC and Figures 4-27, 4-28, and 4-29 are bar graphs depicting the relative 
amount of trkC active and truncated receptors, respectively. Figure 4-30 is the 
phosphorimaging view of the standard for these experiments, cyclophilin. The fact that 
gene expression in these animals is unchanged is surprising in light of the fact that the 
gross state of the nervous system-as evidenced by brain weight and brain weight to body 
weight ratio-was greatly affected by EPET. Since the BGS-with the hippocampus 
sustaining significant neuronal loss-has proven to be a sensitive period for ethanol 
exposure (Bonthius and West, 1991; West et al., 1986), more research will be necessary to 
determine if perhaps changes in other NTFs are responsible for EPET damage to this 
system. 



Figure 4-19. Phosphorimaging view of BDNF Northern blots from postnatally exposed 
P21 rats. Images are obtained from Northern blots composed of male and female animals. 
Each sex is broken down further into Unseparated, Separated, and Ethanol groups as 
described previously in the methods section of this chapter. Two transcripts for BDNF 
were detected. The larger transcript is known to be 4.4 kb and the smaller one is known to 
be 1.7 kb in length. These sizes were confirmed by comparing the transcripts to the 
darkest band of the ladder. Number of lanes used in the analysis equal to 6 for each group. 



118 



Male 



Female 



Unseparated Separated Ethanol 



Unseparated Separated Ethanol Ladder 




^.pr 



*>».f.KJ^ 



f-'^fm 



4.4 kb 



1.7 kb 




m 



vi 



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Figure 4-19. 



119 



0.02 




Ethanol Separated Unseparated 

Figure 4-20. Relative BDNF 4.4 kb transcript gene expression following 
postnatal exposure in P21 rats. A. Female animals. B. Male animals. Ethanol, 
Separated, and Unseparated animals are as described earlier in the Methods 
section of this chapter. Measurements are means + SEM. Analysis of variance 
indicated no significant differences among the groups. n=6 for each group. 



120 



0.0? 




Ethanol Separated Unseparated 
Figure 4-21. Relative BDNF 1.7 kb transcript gene expression following 
postnatal exposure in P21 rats. A. Female animals. B. Male animals. Ethanol 
Separated, and Unseparated animals are as descnbed earlier in the Methods 
section of this chapter. Measurements are means + SEM. Analysis of variance 
indicated no significant differences among the groups. n=6 for each group 



Figure 4-22. Phosphorimaging view of NT-3 Northern blots from postnatally exposed 

P21 rats. Images are obtained from Northern blots composed of male and female animals. [ 

Each sex is broken down further into Unseparated, Separated, and Ethanol groups as j 

described previously m the methods section of this chapter. The NT-3 transcript is known | 

to be 1.5 kb in length. This size was confirmed by comparing the distance relative to the f 

darkest band of the ladder. Number of lanes used in the analysis equal to 6 for each group. j 



122 



Male 



Female 



Unseparated Separated Ethanol 



m 




Unseparated Separated Ethanol 



Ladder 















?v#pfj3p;f^-g?gi&' 









m 






v^sSS* 







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l.Skb 








Figure 4-22. 



123 



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0.02 



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X 


5- 


w 


O 


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Hi 


fl 




<u 




o 






Ethanol Separated Unseparated 



Figure 4-23. Relative NT-3 1.5 kb gene expression following postnatal exposure 
inP21rats. A. Female animals. B. Male animals. Ethanol, Separated, and 
Unseparated animals are as described earlier in the Methods section of this 
chapter. Measurements are means + SEM. Analysis of variance indicated no 
significant differences among the groups. n=6 for each group. 



124 



0.12 




Ethanol Separated Unseparated 



Figure 4-24. Relative trkB active receptor gene expression following postnatal 
exposure in P21 rats. A. Female animals. B. Male animals. Ethanol, Separated, 
and Unseparated animals are as described earlier in the Methods section of this 
chapter. Measurements are means + SEM. Analysis of variance indicated no 
significant differences among the groups. n=6 for each group. 



125 



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tin 


u 


m 


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a> 




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Ethanol Separated Unseparated 



Figure 4-25. Relative trkB truncated transcript gene expression following 
postnatal exposure in P21 rats. A. Female animals. B. Male animals. Ethanol, 
Separated, and Unseparated animals are as described earlier in the Methods 
section of this chapter. Analysis of variance indicated no significant differences 
among the groups. n=6 for each group. 



Figure 4-26. Phosphorimaging view of trkC Northern blots from postnatally exposed P21 
rats. Images are obtained from Northern blots composed of male and female animals. 
Each sex is broken down further into Unseparated, Separated, and Ethanol groups as 
described previously in the methods section of this chapter. The largest band corresponds 
to the active form of trkC (14 kb). The two smaller bands are truncated versions of trkC 
(4.7 kb and 3.9 kb). Size was confirmed by comparing the bands relative to the darkest 
band on the ladder. Number of lanes used in the analysis equal to 6 for each group. 



127 



Male 



Female 



Unseparated Separated ICthanol 



• -* "■•- 



Unseparated Separated l<:thanol Ladder 



:^-: 



^•<y. 






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J- 



14 kb 



■ 3 



M 






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4.7 kb 
3.9 kb 




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Figure 4-26. 



128 



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w 


o 

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0.01 


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Ethanol Separated Unseparated 



Figure 4-27. Relative trkC 14 kb transcript gene expression in P21 rats following 
postnatal exposure A. Female animals. B. Male animals. Ethanol, Separated, 
and Unsepara.ted animals are as described earlier in the Methods section of this 
chapter. Measurements are means + SEM. Analysis of variance indicated no 
significant differences among the groups. n=6 for each group. 



129 



0.06 















0.06 


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0.02- 


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Ethanol Separated Unseparated 



Figure 4-28. Relative trkC 4.7 kb truncated transcript gene expression in P21 rats 
following postnatal exposure. A. Female animals. B. Male animals. Ethanol, 
Separated, and Unseparated animals are as described earlier in the Methods 
section of this chapter. Measurements are means + SEM. Analysis of variance 
indicated no significant differences among the groups. n=6 for each group. 



130 



A 0.04 



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001 


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0- 



Ethanol Separated Unseparated 



Figure 4-29. Relative trkC 3.9 kb truncated transcript gene expressionin P21 rats 
following postnatal exposure. A. Female animals. B. Male animals. Ethanol, 
Separated, and Unseparated animals are as described earlier in the Methods 
section of this chapter. Measurements are means + SEM. Analysis of variance 
indicated no significant differences among the groups. n=6 for each group. 



Figure 4-30. Phosphorimaging view of cyclophilin Northern blots from postnatally 
exposed P21 rats. Images are obtained from Northern blots composed of male and female 
animals. Each sex is broken down further into Unseparated, Separated, and Ethanol 
groups as described previously in the methods section of this chapter. The transcript is 
approximately 1.0 kb in size relative to the darkest band. Number of lanes used in the 
analysis equal to 6 for each group. 



132 



Male 



Female 



Unseparated Separated Ethanol 



Unseparated Separated Ethanol Ladder 





■. i." 






.^\'-9± 

















Figure 4-30. 



133 



Discussion 
CPET 



The results showed that the overall growth of male and female animals was not 
affected by prenatal ethanol exposure. Neither male nor female animals had decreased 
body weight at P21 following CPET. Previous studies in this laboratory have found that 
CPET produces a decrease in body weight at birth that does not persist through P21 
(Swanson et al., 1995; Swanson et al., 1996). The current results agree with these 
findings. The fact that body weight and crown-rump length were not affected by CPET 
suggests that there were not any nutritional differences among the various groups. The 
liquid diet used in this study was developed to provide extra nutritional benefits to 
counteract any possible deficiencies that ethanol might cause. The growth of the nervous 
system-as evidenced by brain weight to body weight ratio-was unaffected by CPET. 
Previous studies from this laboratory did not find that brain weight differences persist until 
P21 (Swanson et al., 1995; Swanson et al., 1996). Also, ethanol was administered at the 
same level in both studies and rats from the same breeding colony were used (Swanson et 
al., 1995; Swanson et al., 1996). Changes, or the lack of a significant change, in brain 
weight did not correlate with changes, or a lack of a change, in gene expression in the 
genes examined in the present study. Specifically, male animals exposed to ethanol 
prenatally did not exhibit a significant change in brain weight or brain to body weight ratio. 
This group was the only one found to exhibit a change in gene expression, with trkC being 
significantly reduced. Conversely, the significant decrease in brain weight found in female 
animals did not correlate with a change in expression of neurotrophin or neurotrophin 
receptor genes studied presently. Overall, these results suggest that ethanol has the ability 
to affect neurons at the fundamental level of gene expression. 

The only significant change in gene expression found in the present study following 
ethanol exposure was in trkC expression in male animals following CPET. Further 
analysis found that trkC expression was affected in more than one way. In addition to the 



134 

absolute decrease in trkC active receptor transcript, the ratio of active receptor transcript to 
truncated receptor transcript was also decreased following CPET. This latter compaiison is 
significant using the PLSD analysis, but not significant following the Bonferroni/Dunn 
correction. The difference between full length and truncated trkC receptors is important to 
this study. A full length trkC receptor is capable of transducing a signal from NT -3 that 
has the ability to alter many processes in the cell, including the activity of proteins capable 
of regulating genetic transcription. A truncated receptor is like a full length receptor, except 
that it lacks a catalytic domain capable of transducing the signal. Developmentally, 
truncated receptors are expressed later than their full length counterparts. By adulthood, 
truncated receptors become the dominant form (Escandon et al., 1994). The implication of 
this change in the ratio of truncated to active receptor is that hippocampal neurons might be 
less able to respond to NT-3 in their environment. The ratio of truncated to active receptors 
may have important implications for neuronal survival and normal neuronal function. The 
possibility exists that the truncated receptors are a dominant negative effect on trk signaling. 
That is, the truncated receptors might inhibit the overall function of trk receptors by either 
binding NT-3 or associating with active trk receptors. Recall that NT-3 has the ability to 
bind trkB and trkA receptors, but with less affinity than to trkC. Thus, NT-3 does not stay 
bound as long to these other receptors as it does to trkC. It is also important to note that no 
other neurotrophins have the ability to bind to trkC. The most efficient signal that NT-3 
produces would occur from binding to trkC. The greater amount of truncated trkC in 
comparison to active trkC also suggests that a given NT-3 molecule would be more likely 
to encounter a truncated receptor than in a control animal. Thus, a similar amount of NT-3 
in the cellular environment would produce less of a signal in these ethanol-treated animals 
than in control animals. 

The role that the low-affinity neurotrophin receptor, p75, may play in ethanol 
neurotoxicity should also be discussed. There is evidence that unbound p75 receptor may 
induce apoptosis in neurons. That is, p75 that is not associated with a trk receptor may 



135 

promote cell death (Kaplan and Miller, 1997). This idea is supported by the fact that NGF 
promotes apoptosis in retinal neurons of the chick that express p75, but do not express 
trkA (Frade et al., 1996). Furthermore, mice that express the intracellular domain of p75 
exhibit widespread CNS and PNS neuronal loss (Majdan et al., 1997). In fact, the level of 
cell death induced by p75 seems to be directly proportional to the amount of p75 expressed. 
Higher expression of p75 correlated with higher levels of apoptosis in cultured 
neuroblastoma cells (Bunone et al., 1997). All of this evidence supports the idea that 
unbound p75 may induce cell death in neurons. Since the present study found a reduction 
in trkC gene expression, it might be logical to assume that such a reduction would result in 
increased levels of unbound p75. As a result, more hippocampal neurons would perish. 
The knockout studies do not necessarily support this claim, since NT -3 knockouts are 
phenotypically more impaired than trkC knockouts (Conover and Yancopoulos, 1997). 
Careful examination of hippocampal pyramidal cell number in knockout animals has not yet 
been completed to date. As was mentioned above, few CNS populations are reduced 
following neurotrophin and neurotrophin receptor gene deletion. The fact that CNS 
populations are not greatly affected could be due to changes in the genetic background of 
the knockout animals and not due to the true loss of activity of the neurotrophin or 
neurotrophin receptor of interest. 

NT -3 expression remains high throughout CNS and hippocampal development 
(Maisonpierre et al., 1990). The fact that its expression is so high suggests that it is 
important for neuronal development during this time period. Therefore, a change in the 
ability of hippocampal neurons to detect the NT -3 signal due to a reduction in available trkC 
receptors could alter the survival status, or some other general status of the cell. Since 
pyramidal cells that perish due to ethanol exposure are most likely already dead by P21, it 
is possible that the observed reduction (P21) in trkC gene expression has little effect on 
neuronal loss in the hippocampus. As learning and memory are impaired by developmental 
ethanol exposure (Abel, 1995; Jones and Smith, 1973; Streissguth et al., 1991), and 



136 

learning and memory are controlled by living neurons, it may be that those neurons that 
remain do not perform their functions at a normal level. NTFs and NTF receptors have 
been linked to long-term potentiation (LTP) in the hippocampus (Bramham et al., 1996; 
Kang and Schuman, 1995; Kang and Schuman, 1996). LTP has been postulated as part of 
the mechanism for encoding new memories (Bunsey and Eichenbaum, 1996). Therefore, a 
reduction in trkC could reduce the ability of hippocampal neurons to carry out LTP and 
form new memories. This relationship is important to the current study because our 
findings suggest that the ability of developing hippocampal granule and pyramidal neurons 
to detect this important NTF might be significantly impaired as a result of prenatal ethanol 
exposure. 

Pyramidal neurons--a population known to express trkC (Chao and McEwen, 
1994)--are greatly affected by prenatal ethanol exposure (Barnes and Walker, 1981). It is 
possible that the reduction in this population is responsible for the reduction in trkC 
expression observed in the present study. It is also important to examine why BDNF, NT- 
3, and trkB gene expression are unaffected by ethanol exposure even though there is a 
significant loss of cells expressing these proteins in the hippocampus (Barnes and Walker, 
1981). Granule cells, pyramidal cells, and glia of the hippocampus all express BDNF, 
NT-3, trkB, and trkC (Chao and McEwen, 1994; Condorelli et al., 1995; Dragunow et al., 
1997; Mathem et al., 1997). Using pyramidal cell loss as a potential explanation for the 
reduction in trkC expression is refuted by the fact that all NTF and NTF receptor genes 
analyzed in this study were standardized by a constituitively-expressed housekeeping gene. 
That is, the reduction in pyramidal cells should not selectively reduce trkC since all other 
cell types in the region also express trkC. Also, a loss of pyramidal neurons that express 
trkC would also result in a loss of an equal number of cyclophilin-expressing neurons. 
Therefore, the reduction of trkC observed in our study is a reduction per unit volume of 
tissue and not just a gross subtraction due to cell loss. 



137 

The results of the present study also relate to a previous study that found that 
ethanol reduces neurofilament levels in cultured hippocampal neurons (Saunders et al., 
1997). Neurofilaments are necessary for neurite outgrowth (Saunders et al., 1997). Since 
neurotrophins promote neurite outgrowth, presumably through their interaction with trk 
receptors, the effect observed in the Saunders et al. study could be due to a reduction in 
trkC expression (1997). The reduction in trkC would likely result in decreased NT-3 
signal. Less neurofilament protein would be produced and as a result, and fewer neurites 
would be extended, which would then affect the individual neurons ability to make 
synapses and receive neurotrophic support. The results of the present study may explain 
the results of some previous studies and may help to answer questions about the 
fundamental nature of ethanol toxicity. 
EPET 

There were more physical differences at P21 following EPET than there were 
following CPET. As evidenced by gross body measurements, male animals were affected 
to a greater extent by EPET than female animals. Male Ethanol animals actually weighed 
more than both Separated and Unseparated animals at P21 (refer to Figure 4-3). This 
finding is probably due to the culling procedure employed in this study (ethanol litters were 
limited to seven pups and control litters had 10 pups). Following the exposure period, 
which ended on PIO, the Ethanol pups had greater opportunity to nurse than the control 
pups did and gained more weight. Both male and female animals had decreased brain 
weight following EPET. This difference was further borne out by the fact that brain weight 
to body weight ratio was reduced in both male and female Ethanol animals in comparison to 
I both Separated and Unseparated animals. The physical differences described above did not 

result in a change in the gene expression of any of the genes analyzed in this study. 

The fact that gene expression was unchanged in BDNF, NT-3, trkB and trkC 
following EPET could indicate that earlier observations of decreased neurotrophic activity 
following EPET were due to an NTF other than one of the neurotrophins studied. Other 



] 



138 

NTFs, such as bFGF, are produced in the hippocampus and are important to the normal 
functioning of neurons in this brain region (Ernfors et al., 1990). Also different genes are 
more or less active at different points during development (Maisonpierre et al, 1990). 
Therefore, it is possible that EPET affects other genes or may affect the genes studied to an 
extent that is not detectable by methods used in the present study. 

General Discussion 

Taken together all of the results suggest that ethanol does have the ability to regulate 
NTF receptor gene expression to some extent. Specifically, trkC gene expression was 
significantly lower in male rats following CPET than in control animals. This study is not 
the first to find that ethanol has the ability to alter gene expression. Prenatally, ethanol 
reduces IGF-I (Breese et al., 1994; Singh et al., 1996) and IGF-II in rat brain (Singh et al., 
1996). In contrast, IGF-II gene expression is increased in fetal lung tissue (Fatayerji et al., 
1996). Ethanol exposure increases c-jun and junD levels in cultured neuroblastoma cells in 
a dose-dependent manner (Ding et al., 1996) and increases NMDA receptor gene 
expression in cultured mouse cortical neurons (Hu et al., 1996). The fact that c-jun is 
affected by ethanol treatment could be could be important. C-jun is an immediate early 
gene and is activated before a change in genetic expression occurs. Immediate early genes 
are thought to control growth and differentiation of neurons by regulating other genes 
(Abraham et al., 1991). Members of the jun family form dimers and bind to DNA to 
regulate transcription (Vogt and Morgan, 1990). It is possible that c-jun and junD may be 
responsible for the change in expression of trkC observed in the present study. If these 
factors regulate trkC expression negatively, increasing their expression would likely cause 
a decrease in trkC gene expression. The fact that ethanol can regulate this gene further 
demonstrates that genetic control in a developing neuron can be affected by ethanol 
treatment. 

Other studies have examined ethanol' s ability to regulate genetic expression 
following development. Chronic ethanol treatment in adult rats resulted in a reduction of 



139 

BDNF gene expression in the hippocampus (MacLennan et al., 1995). This result is in 
contrast to the results of the present study. However, the current study is developmental 
and should not necessarily correlate with adult chronic ethanol treatment studies because of 
the state of the nervous system during ethanol exposure. The developing CNS contains 
many critical periods where functional synapses are being formed and where a slight 
disruption can result in neuronal loss. The adult nervous system, while vulnerable to 
teratogenic effects of ethanol, does not contain an environment in a state of developmental 
flux. Therefore, exposure to ethanol during development-whether in utero or postnatal-is 
different from adult ethanol treatment. Trk gene expression was not analyzed in the 
MacLennan et al. study but has been analyzed following adult ethanol exposure. 
Specifically, trkB expression was observed to be upregulated following ethanol exposure 
in adult male rats (Back et al., 1996). 

The present experiments were designed to precisely determine whether BDNF, 
NT -3, trkB, or trkC gene expression in the hippocampus is altered as a result of ethanol 
exposure. NGF gene expression was also attempted, but repeated attempts at probing 
failed to yield quantifiable signals. Efforts to obtain results from NGF probing included 
longer exposure to the phosphorimaging plates, increase in the amount of probe, and 
increase in the amount of radioactivity. Following a lack of success with each of these 
potential solutions, NGF probing was abandoned. Previous experiments in our laboratory 
have suggested that ethanol exposure does change the amount of neurotrophic activity 
present in extracts made from rat hippocampus. These experiments involved culturing 
DRG neurons in the presence of these extracts and assessing survival and neurite 
outgrowth. Specifically, CPET increased neurotrophic activity of hippocampal extracts at 
P21 (Heaton et al., 1995c), and EPET decreased neurotrophic activity of hippocampal 
extracts at this same age (Moore et al., 1996). Even though in the present study there was 
no change detected in the expression of the neurotrophins studied, these results do not 
necessarily disagree with those previous studies from this laboratory. Since other NTFs 



140 

are synthesized in the hippocampus, overexpression of another polypeptide could have 
produced the increase in neurotrophic activity observed following CPET. 

The age examined in the present study was chosen because previous studies which 
analyzed the neurotrophic activity following both CPET (Heaton et al., 1995c) and EPET 
ethanol exposure (Moore et al., 1996) both found altered neurotrophic activity at this, and 
not any other, age. Gene expression should be examined at other time points to determine 
the temporal extent of the decrease in trkC expression. Specifically, trkC and other NTF 
and NTF receptor genes should be assessed at time points that are closer to the period of 
ethanol exposure, and at time points between the completion of ethanol exposure and P21. 
Additionally, analysis should also occur at a time point near maturity to determine if any 
affect of developmental ethanol exposure is long-lasting. For prenatally exposed animals 
ethanol exposure ends on PO so the analysis should take place on PI, P7, P14, and P60. 
For postnatally exposed animals ethanol exposure runs from P4 to PIO. Therefore, 
analysis should occur on PIO, P14, and P60. Additionally, analysis during the exposure 
period might reveal ongoing effects of ethanol exposure with respect to gene expression. 
Also, other brain regions known to be affected by ethanol exposure, such as the cerebellum 
(Cragg and Phillips, 1985; West, 1986) and the cerebral cortex (Miller, 1986) should be 
examined to determine if ethanol affects NTF or NTF receptor gene expression. The 
amount of functional NTF and NTF receptor protein present in the hippocampus should 
also be examined. Since a reduction of trkC gene expression does not necessarily correlate 
with a reduction in the functional trkC protein, future studies should examine these animals 
to determine if this is indeed the case. 

The results of the present study may have some bearing on the results of the 
neurotrophic activity following prenatal ethanol exposure study performed in this 
laboratory (Heaton et al., 1995c). Perhaps NTFs were over-produced to compensate for 
the lack of responsiveness in hippocampal neurons due to the decrease in trkC expression. 
Previous research has shown that ethanol exposure causes an upregulation of NGF protein 



141 

in rat hippocampus and cortex (Nakano et al., 1996). Other NTFs and NTF receptors have 
been found to be upregulated following neuronal injury. These include CNTF following 
entorhinal cortex lesion (Lee et al., 1997) and trkA following striatal and basal forebrain 
injury. The decrease in trkC gene expression could also be interpreted as a neuronal 
response to injury. Previous research has shown that when the hippocampus is injured by 
ibotenic acid, what results is a neuron poor, astroglia rich environment (Belluardo et al, 
1995). In this setting, trkC gene expression was significantly reduced. The possibility that 
ethanol produces an injury type of response is interesting, but that fact alone would not 
explain the mechanism by which ethanol could injure the neurons. One candidate 
mechanism for altering gene expression following ethanol exposure is a change in DNA 
methylation. As was mentioned above, DNA is modified by methylation at the 5' position 
of cytosine residues. This process converts cytosine to methylcytosine and is thought to 
interfere with the binding of proteins-repressors or enhancers--to DNA to change gene 
expression (HoUiday, 1987). This change in methylation can either increase or decrease 
the transcription of a given gene and thereby alter the expression since repressors or 
enhancers are not selectively affected. 

The relationship between cell types found in the hippocampus, the neurotrophin and 
neurotrophin receptor genes that they express, and their susceptibility to ethanol may help 
explain the present results. Cell types found in the hippocampus include granule cells, 
pyramidal neurons, and glia. Hippocampal granule cells express NOP (Lindefors et al., 
1992), BDNF (Mathem et al., 1997), NT-3 (Mathem et al., 1997), trkB (Dragunow et al., 
1997), and trkC (Dragunow et al, 1997). Pyramidal neurons are known to express 
BDNF, NT-3, trkB, and trkC (Chao and McEwen, 1994) as well as bFGF (Chao and 
McEwen, 1994; Walicke, 1988) and NGF (Lindefors et al., 1992). Glial cells also express 
some of the NTFs used in the present study. Specifically, microglia, astroglia, and 
oligodendrocytes express NT-3, trkB, and trkC as well as NGF (Condorelli et al., 1995). 
Following prenatal ethanol exposure, pyramidal neuron loss is observed in the 



142 

hippocampus (Barnes and Walker, 1981). Further examination of the pyramidal neurons 
revealed that synapses and dendritic branching were also greatly affected by ethanol 
exposure (Smith and Davies, 1990). 

The effect of ethanol exposure on granule neurons is not as clear. Pierce and West 
(1987) found that granule cells are increased following postnatal ethanol exposure while the 
overall area of the hippocampus was reduced. Another laboratory found a reduction in 
mamre granule neurons following prenatal exposure (Wigal and Amsel, 1990). Based on 
these studies, it appears that granule cells have a specific temporal vulnerability to ethanol 
toxicity. The reduction of trkC gene expression observed in the present study could be due 
to a reduction of any of the hippocampal cellular populations, but the fact that pyramidal 
cells are greatly affected by ethanol exposure suggests these cells might be where the 
largest change is occuixing. However, pyramidal cells that are affected by ethanol 
exposure are most likely already dead by P21. Logically, trkC is probably altered in its 
expression in other cell types. As was mentioned above, the loss of neurons from the 
hippocampus results in a neuron poor, astroglia rich environment, where trkC is reduced in 
expression (Belluardo et al., 1995). A future aim of this research should be to use in situ 
hybridization to determine whether trkC expression is affected in one particular cell type in 
the hippocampus. That is, is trkC gene expression reduced in pyramidal cells preferentially 
over granule cells or glia. Since pyramidal cells are most likely already reduced in number 
by P21, such an analysis should take place over a time course that is close in proximity, 
and perhaps includes, the period of ethanol exposure. Analyzing the expression of trkC in 
situ, and correlating this expression with pyramidal and granule cell counts on the same 
day, would possibly determine if trkC expression in this cell population is related to cell 
loss. The fact that all of the cell types in the hippocampus express trkC is important, 
because it suggests that these populations respond to NT-3. Therefore, a significant 
reduction of trkC could greatly affect these neurons. A relatively simple way to determme 
if trkC is required for survival of pyramidal neurons in vivo is to count surviving neurons 



143 

in knockout animals. To date, this analysis has not been completed, but such a study 
would help to determine if a reduction in trkC causing significant pyramidal cell loss is 
physiologically relevant. 

The fact that the present study found that female animals were not affected in a 
manner similar to male animals in terms of genetic expression by prenatal ethanol exposure 
suggests that there is a fundamental difference between male and female animals' ability to 
withstand ethanol insult. While the concept of sex-dependent effects of ethanol is not new, 
previous research has been somewhat inconclusive in finding an absolute difference in 
susceptibility between male and female animals following ethanol exposure. In addition to 
the present results, our laboratory has identified gender differences in the effect that ethanol 
had on neurotrophic activity in the hippocampus of postnatally exposed rats (Moore et al., 
1996). Specifically, male animals were affected to a greater degree than females. The 
hippocampus has been previously shown to be affected in a sexually dependent manner in 
that male rats exhibit a spatial learning deficit that is not found in female animals following 
prenatal exposure (Zimmerberg et al., 1991). The interesting result among these studies is 
that male animals are affected to a greater degree than female animals in all instances. In 
fact, the studies that initially observed pyramidal neuron loss in the hippocampus were 
performed using only male animals (Barnes and Walker, 1981; West, 1986). The 
implication is that the hippocampus in male animals may be more susceptible to ethanol 
insult than it is in female animals. This result-hippocampal impairment— is logical 
considering that learning and memory deficits are a common characteristic of FAS (Abel, 
1995; Jones and Smith, 1973; Streissguth et al., 1991). The fact that normal hippocampal 
functioning is impaired by ethanol treatment may explain the behavioral deficits observed in 
FAS. That is, impairment of the septohippocampal system by ethanol is a possible cause 
of these behavioral deficiencies. 

Other than the hippocampus, brain regions affected in a sexually dependent manner 
include the septum and the amygdala. Our laboratory found that female rats exposed to 



144 I 



ethanol exhibit a greater reduction in septal parvalbumin neurons than their male 
counterparts (Moore et al., 1997) and prenatally-exposed male rats contain greater numbers 
of cholinergic septal neurons than female ethanol-exposed animals (Swanson et al., 1996). 
Thus, both septal studies exhibit a greater effect among female animals. Other laboratories 
have discovered gender differences in response to ethanol exposure. DNA production is 
significantly reduced in the amygdala of male rats, but unchanged in female animals 
following prenatal ethanol exposure (Kelly and Dillingham, 1994). On a macro level male 
and female animals respond differently to stress following ethanol exposure in that ethanol- 
fed females exhibited increased corticosterone concentrations in comparison to ethanol-fed 
males (Giberson et al., 1997; Weinberg, 1992). All of these studies show that ethanol can 
affect the sexes differently, but not in a consistent manner. One sex is not preferentially 
affected in all instances. The fact that sexual differences are so common underlies the 
importance of analyzing sexes separately in ethanol exposure studies. 

Other studies that have examined possible gender disparities did not find significant 
disparities. For example, the locus coeruleus was affected by ethanol treatment, but no 
gender difference was observed (Lu et al., 1997). Alcohol dehydrogenase activity was 
found to exhibit no gender differences in a variety of mouse strains (Rao et al., 1997). Our 
laboratory found that ChAT activity was not altered in a sexually dependent manner in male 
and female animals following ethanol exposure (Swanson et al., 1995). Even though 
different sexual responses to ethanol are not present in all studies, the possibility that 
ethanol may differently affect male and female animals should continue to be explored 
further in future experiments. 

The process of exploring the phenomenon of ethanol-NTF interaction in the 
nervous system would require the use of tissue culture to investigate signal transduction in 
hippocampal cultures. The cultures would have to be grown in serum-free media since 
serum contains large amounts of undefined proteins and would greatly complicate analysis. 
Large cultures ( 10^ in number or greater) of hippocampal neurons necessary to study signal 



145 

transduction can be obtained. However, the PC 12 immortalized cell line has been used 
extensively in studying signal transduction of trk receptors (Stephens et al., 1994). Even 
though PC 12 cells have been widely used to study trk signaling, primary hippocampal 
cultures would be preferable for this analysis because they are normal neuronal cells and 
not merely a model. While the tissue culture environment does lack some of the in vivo 
interactions that are important for a complete analysis of any interaction between trk signal 
transduction and ethanol--such as glial cell/neuron interactions-it is the only method that 
allows specific signaling events to be studied. NT-3 would be the logical NTF to use in 
studying these events since its receptor, trkC, is affected by CPET and expressed by this 
neuronal population (Chao and McEwen, 1994; Dragunow et al., 1997). While this 
analysis would not explain the decrease in trkC gene expression observed following 
prenatal ethanol exposure, it would help to further define the role of ethanol toxicity as it 
relates to NTFs. This relationship connects all of the studies in this overall document. 

The present study investigated the ability of ethanol to regulate BDNF, NT-3, trkB, 
and trkC gene expression. Since trkA is not expressed in the hippocampus it was not 
examined in this study (Martin-Zanca et al, 1990). NGF, while expressed at a level that is 
above the threshold of detection for Northern blotting (Maisonpierre et al., 1990), did not 
yield bands that could be quantified. Perhaps a future goal of this study should be to 
examine NGF expression with a finer molecular biology technique-such as RNAse 
protection assay. Overall, the results of this study indicate that ethanol can alter the 
expression of NTF receptor genes when administered prenatally. The results of the present 
study should serve as a point of reference in the search for a mechanism of ethanol toxicity. 
Since trkC is now known to be regulated by CPET, future researchers should determine 
exactly how this gene is affected by ethanol. 



CHAPTERS 
CONCLUSIONS AND IMPLICATIONS 

Animal Models 
The research described in this document was undertaken to describe the relationship 
between ethanol and neurotrophic factors (NTFs) in the developmg nervous system. Two 
animal models were used to carry out this objective. The chick embryo model was used to 
determine the effect that ethanol had on the developing motoneuron population of the spinal 
cord and to test the ability of NTFs to modulate the toxicity of ethanol in vivo. The rat 
animal model was used to determine the effect that ethanol treatment during hippocampal 
development had on NTF and NTF receptor gene expression. Each animal model was 
selected for a specific advantage it had in completing the proposed studies. The chick was 
chosen because ethanol can be administered in exact doses to the developing embryo, and 
only molecules produced by the embryo itself remove ethanol from the embryonic 
environment. Ethanol is cleared from the bloodstream by the mother in a mammalian 
system whereas the chick embryo is isolated as it develops. The fact that maternal 
influences are removed does not make the chick ideal for comparisons to human fetal 
alcohol syndrome (FAS), but does allow for the examination of direct effects of ethanol on 
a developing neuronal population. Additionally, the chick embryo model has been widely 
used to study the effects of NTFs on the developing motor system (Oppenheim et al., 
1995; Oppenheim et al., 1992). NTFs can be administered through small holes directly to 
the embryo onto the chorioallantoic membrane since the embryos tolerate slight mvasions 
into their environment quite well as long as the underlying membranes are not disrupted. 

The rat was chosen to examine the ability of ethanol to alter gene expression of 
NTFs because it is a mammal and ethanol exposure would be similar to that found in 



146 



147 



humans. Additionally, the rat is the most widely studied model of FAS research and 
exhibits many of the same deficits found in human FAS (Diaz and Samson, 1980; Sherwm 
et al., 1981). As a mammal, the rat central nervous system (CNS) compares favorably 
with the human and contains the same major structures found in the human CNS. 
Additionally, our laboratory has found that prenatal exposure to ethanol increased 
neurotrophic activity of hippocampal extracts on postnatal day 21 while postnatal exposure 
to ethanol decreased neurotrophic activity of these same extracts (Heaton et al., 1995c; 
Moore et al., 1996). The rat has also been used in previous research which found that 
chronic ethanol treatment in adult male rats resulted in a decrease in the expression of the 
brain-derived neurotrophic factor (BDNF) gene (MacLennan et al., 1995). The adult 
exposure utilized in that experiment is different from the prenatal exposure used in the 
present study in that adult exposure requires only the subject's liver to remove ethanol from 
the bloodstream. In prenatal exposure, the mother's liver can remove ethanol from the 
bloodstream. Eariy postnatal exposure in rats, even though the nervous system is 
developmentally like a prenatal human nervous system, is like adult exposure. However, 
the CNS environment is very different in developing and adult animals. Where the adult 
CNS is relatively stable, the developing CNS is undergoing rapid changes that will 
ultimately lead to the structures of the adult CNS. During this active stage of development, 
the CNS is also susceptible to disruptions that would not normally harm an adult CNS. 
Teratogens that act on these neurons can have a long-lasting effect on the development of 
the CNS. The studies described previously are related in that they all examine some facet 
of ethanol toxicity as it relates to NTFs. The studies examined two sides of one issue: 

Methods 
The methodology employed with each animal model in the present research was 
chosen for specific reasons. Since the present research builds on results from previous 
work-both from this laboratory and from outside laboratories-the methods used in this 
study were similar to these previous studies. By replicating methods that had been used in 



148 

the past, better comparisons could be made between our results and previous work. For 
example, the method for delivering NTFs to embryonic chicks was similar to that used in 
studies from Oppenheim's laboratory (Oppenheim et al., 1995; Oppenheim et al., 1992). 
Delivery of ethanol to prenatal and postnatal rats replicated methods used in studies from 
our laboratory (Heaton et al., 1995c; Heaton et al., 1996; Moore et al., 1996; Ryabinm et 
al., 1995; Swanson et al., 1995; Swanson et al., 1996). Northern blotting techniques were 
similar to those used in assessing the effect chronic ethanol treatment had on NTF gene 
expression in the adult hippocampus (MacLennan et al., 1995). Thus, it is clear that great 
care was taken to ensure that valid comparisons were made between the present studies and 
previous ones. 

The method used to deliver NTFs to developing chick embryos was ideal in that the 
developing embryo was never physically contacted during the procedure. Past experience 
in this laboratory has found that an embryo will likely die if the membranes are disturbed. 
One disadvantage of our method was that an occasional embryo was lost because of human 
error with the injecting procedure. Another disadvantage is that NTFs are administered 
systemically and almost certainly exert effects in addition to those described in Chapter 3. 
Future experiments will have to determine whether the current evidence that GDNF 
increases motoneuron number in a manner independent to ethanol is achieved by protection 
of developing motoneurons from ethanol toxicity. The current experiments do not allow a 
conclusion of neuroprotection from ethanol toxicity to be drawn. Such an analysis would 
require an interaction between ethanol and GDNF. Specifically, experiments will have to 
detennine whether ethanol directly hinders the toxic mechanism of ethanol in the nervous 
system or some other independent mechanism. For example, ethanol is known to affect 
Ca^"" homeostasis in cultured neurons (Myers et al., 1984; Webb et al., 1995). The next 
step would be to determine whether GDNF can directly prevent such a change from 
occurring. In the current study, NTFs were administered through pinholes in the eggshell 
and dropped onto the chorioallantoic membrane of developing chick embryos. The fact that 



149 

survival was highest in animals receiving an NTF only suggests that NTF administration 
had little adverse effect on embryonic survival. The methods used to deliver ethanol to 
developing rats have been used previously m this laboratory and in others. The ethanol- 
containing liquid diet fed to pregnant dams has been shown to produce deficits in the 
hippocampus while providmg nutrition above and beyond control conditions (Barnes and 
Walker, 1981). Ethanol inhalation was used to build on a previous study from this 
laboratory and because it has been shown to be an effective, nonintrusive method for 
delivering ethanol to neonatal rats (Moore et al., 1996; Ryabinin et al., 1995). While 
artificial rearing is also effective in delivering precise doses of ethanol to the subjects, the 
possible stresses introduced by this procedure can make interpretation of the results 
difficult. For these reasons, and those discussed in Chapter 4, ethanol inhalation was used 
to simulate ethanol exposure during the brain growth spurt. 

Quantitative Northern blotting was used to determine the relative genetic expression 
of neurotrophin and neurotrophin receptor mRNA in the hippocampus. Other methods for 
detecting mRNA expression, such as RNAse protection assay and reverse transcriptase 
polymerase chain reaction, can detect mRNA expression at least about ten times greater 
sensitivity (Lee and Costlow, 1987; Sperisen et al., 1992). The variability found when 
using these methods is often much greater than that found with Northern blotting. When 
genes are readily detected by Northern blotting, analysis of their expression can be 
accurately quantified. All of the genes examined in the current study-BDNF, 
neurotrophin-3 (NT-3), tyrosine receptor kinase (trk) B, and trkC-are expressed at levels 
which make quantitative Northern blotting appropriate and preferable for these experiments 
(Maisonpierre et al., 1990). Originally, our aim was to probe for NGF in addition to the 
above NTFs and receptors. However, repeated attempts at probing for NGF produced no 
quantifiable results. The fact remains that NGF should be expressed at a high enough level 
in the hippocampus to be detected by Northern blotting (Maisonpierre et al., 1990). 
Perhaps the NGF cDNA probe used in these studies does not share enough homology with 



150 

Long Evans rats to stay bound through the high stringency washes. An NGF cDNA probe 
isolated from a rat from our colony might produce quantifiable bands. 

Hypotheses and Results 

The research chapters of this document described all of the experiments performed 
for this study. Each chapter attempted to answer a portion of the hypotheses described in 
Chapter 1: (1) (a) We hypothesized that ethanol would reduce motoneuron number in the 
absence of naturally occurring cell death (NOCD); (b) We hypothesized that ethanol would 
reduce motoneuron number at period of development that follows the period for NOCD; (2) 
We hypothesized that exogenous NTFs would provide in vivo protection for motoneurons 
exposed to ethanol; and (3) We hypothesized that chronic prenatal ethanol treatment 
(CPET) and early postnatal ethanol treatment (EPET) would alter the gene expression of 
neurotrophins and/or their receptors in the hippocampus of treated rat pups. 

The major findings of Chapter 2 were that ethanol did not exacerbate NOCD in the 
developing spinal cord when administered from E4 to Ell; ethanol reduced motoneuron 
number when administered from ElO to E15; and ethanol did not affect the neurotrophic 
content of muscle when administered from ElO to E15. Thus, experiments described in 
Chapter 2 confirmed the hypothesis that ethanol would reduce motoneuron number in the 
lumbar spinal cord when NOCD was suspended and when administered from ElO to E15. 
These experiments provided other information that was valuable in describing the action of 
ethanol upon developing motoneurons. The fact that neurotrophic content of limb muscle 
was unchanged suggests that ethanol was not merely altering the amount of NTFs 
produced by the motoneuronal targets to reduce their number at this age. However, the 
current results do not definitively demonstrate that NTF activity in chick muscle tissue is 
unaffected by ethanol exposure from ElO to E15. The major finding of Chapter 2 was that 
ethanol can act directly to affect this population. A specific way that ethanol could directly 
affect motoneurons is alteration of Ca^"^ homeostasis. Changes in Ca^"" levels are linked to 
neuronal death (Choi, 1988) and ethanol is known to modulate this delicate system (Gandhi 



151 

and Ross, 1989; Leslie et al., 1990; Reynolds et al., 1992; Webb et al., 1995). However, 
these studies do not demonstrate a causal relationship between ethanol and Ca^'^-induced 
cell death. 

Another possible way ethanol could directly harm neurons is by altering membrane 
fluidity. Indeed, ethanol does have the ability to change this important cellular property 
(Avdulov et al., 1995; Schroeder et al., 1988; Wood et al., 1989). If the integrity of the 
membrane is compromised, a cell is susceptible to changes that could result in death. One 
such change could be an alteration of the intracellular ion concentration, since changes in 
membrane fluidization can lead to altered activity in the Na^/K"" ATPase (Madsen et al., 
1992). Furthermore, neurons are dependent on proper concentrations of Na"" and K" to 
generate action potentials. Proper synapse formation is dependent on the ability of neurons 
to generate activity and access to target-derived NTFs is related to synapse formation (Lu 
and Figurov, 1997). Since NTFs promote neuronal survival and maintenance, changes in 
membrane fluidization may have far-reaching effects on the survival of developing 
neurons. However, there is no evidence that NTFs affect membrane fluidity. Therefore, 
experiments that test the ability of NTFs to stabilize or destabilize the cellular membrane 
should be perfomied before attempting to determine whether they can prevent ethanol 
disturbing this integral system. Certainly, other possible mechanisms for ethanol toxicity 
exist and this brief discussion should not be considered to be comprehensive. 

Chapter 3 sought to determine whether motoneuron death due to ethanol in a living 
organism could be prevented by NTF treatment. The major result of this chapter was that 
GDNF significantly increased motoneuron number in the presence of ethanol. BDNF did 
not significantly protect developing motoneurons and did not significantly interact with 
ethanol. Thus our hypothesis that GDNF and BDNF would provide in vivo 
neuroprotection was not confirmed since neither NTF interacted with ethanol. Previously, 
NGF was previously shown to protect against ethanol induced decrease in cholinergic 
activity in whole chick (Brodie et al., 1991). GDNF has been shown previously to provide 



152 

protection against neurotoxic insults. For example, GDNF protects against 6- 
hydroxydopamme lesion in the substantia nigra of the rat (Kearns and Gash, 1995) and 
protects against ischemia induced mjury in rat cortex (Wang et al., 1997). In fact, GDNF 
has even been shown to provide protection against ethanol insult in culture. McAlhany et 
al. found that GDNF rescued rat organotypic cultures of cerebellar Purkinje cells from 
ethanol neurotoxicity (1997). 

Above, two possible mechanisms of direct toxicity were explored and tied to 
ethanol. NTFs are known to affect some of these same processes and at the same time 
promote cell survival. For example, NGF, bFGF, and insulin-like growth factors I and 11 
(IGF-I and IGF-II) were all shown to prevent neuronal death due to excitotoxicity 
presumably by keeping intracellular calcium levels in the cell at sublethal levels (Mattson 
and Cheng, 1993). All of these NTFs are members of different NTF families. 
Specifically, NGF is a member of the neurotrophin family; bFGF is a member of the 
fibroblast growth factor family; and IGF-I and IGF-II are members of the insulin-like 
growth factor family. Thus, NTFs from varied families exhibit similar traits of promoting 
neuronal survival. GDNF, as a member of the transforming growth factor B family, might 
also exhibit these characteristics. To date GDNF's ability to regulate intracellular calcium 
has not been investigated. Therefore, given that ethanol can modulate Ca^"" concentration 
and NTFs can stabilize Ca^^ GDNF might protect motoneurons by holding Ca^-" at a safe 
level. It is important to note that evidence from the current experiments does not suggest an 
interaction between ethanol and GDNF. A relatively simple way to test this hypothesis 
would be to replicate the above experiments to determine whether GDNF can alter Ca^'' 
concentration. 

The experiments described in Chapter 4 used another method of defining the 
relationship between ethanol and NTFs in the developing nervous system to determine 
whether ethanol could modulate the genetic expression of neurotrophins, or their receptors, 
in the hippocampus. The major findings of Chapter 4 were that CPET in the rat reduced 



153 

trkC gene expression in male rat hippocampus on P21. The results both confirm and refute 
the hypothesis for this chapter in that CPET altered genetic expression of trkC while 
postnatal exposure did not alter the expression of any of the genes studied. None of the 
neurotrophin genes were altered following prenatal or postnatal exposure. Therefore, that 
part of the hypothesis was also refuted by the results of the study. This study is not the 
first to demonstrate a change in the in vivo expression of a NTF gene following embryonic 
ethanol exposure. Earlier research found that IGF expression was increased in whole rat 
brain following prenatal ethanol exposure (Breese et al., 1994; Singh et al., 1996). 
However, receptor gene expression was not affected in that study. 

Also important was the fact that female and male animals were not similarly affected 
by ethanol exposure. This result suggests that there is a fundamental difference between 
male and female animals' ability to withstand ethanol insult. The hippocampus appears to 
be more susceptible to ethanol insult in male rats in comparison to female animals. 
Specifically, male rats exhibit deficiencies in spatial learning following prenatal ethanol 
exposure (Zimmerberg et al., 1991), decreased neurotrophic activity following postnatal 
exposure (Moore et al., 1996), and the present findings indicate that prenatal exposure 
reduces trkC gene expression. Other brain regions affected in a sex dependent manner 
include the septum and the amygdala. Specifically, female rats exposed to ethanol 
prenatally exhibit a greater reduction in septal parvalbumin neurons than their male 
counterparts (Moore et al., 1997) and prenatally-exposed male rats contain greater numbers 
of cholinergic septal neurons than female ethanol-exposed animals (Swanson et al., 1996). 
Both studies of the septum display a greater effect of prenatal ethanol exposure on female 
animals. In the amygdala, DNA production is reduced in male animals but unchanged in 
female animals following prenatal ethanol exposure (Kelly and Dillingham, 1994). These 
studies demonstrate the importance of isolating gender in FAS research. 



154 



Conclusions 



One finding of this research was that genetic expression of trkC is reduced 
following prenatal exposure to ethanol in rats. Genes that are known to be affected by 
ethanol treatment include BDNF (MacLennan et al., 1995), IGF-I and IGF-II (Breese et 
al, 1994; Singh et al., 1996), c-jun and junD (Ding et al., 1996), and NMDA receptor (Hu 
et al., 1996). In the current experiments, no neurotrophin genes were altered as a result of 
prenatal or postnatal ethanol treatment. The fact that the jun family is modulated by ethanol 
treatment may help to illuminate a mechanism for ethanol toxicity in the developing nervous 
system. Since c-jun binds DNA directly to regulate transcription, it is possible that ethanol 
alterations in its activity serve to change the expression of other genes. This relationship 
should be explored further in future experiments. 

Since both BDNF and trkC gene expression are known to be affected by ethanol 
exposure (MacLennan et al, 1995), and trk intracellular signaling activates MAP kinase 
(Stephens et al., 1994), it makes sense to examine signal transduction pathways that result 
from trk molecules to determine whether ethanol can interfere. Intracellular trk signaling 
involves phosphorylation of many different proteins and eventually activates proteins that 
are known to alter transcription in the nucleus. Previous research has identified distinct 
pathways involved in trk signaling. Both pathways are initiated by autophosphorylation of 
tyrosine 490 on the intracellular domain of trk (Stephens et al., 1994; Tolkovsky, 1997). 
As mentioned above, one of these pathways leads to activation of the MAP kinase cascade 
and is thought to initiate neurite outgrowth, transcription, or cellular hypertrophy (Stephens 
et al., 1994). The other pathway leads to akt (a serine/threonine kinase) activation and may 
initiate neurite outgrowth, survival, and receptor internalization (Tolkovsky, 1997). Since 
neuronal survival is affected by CPET and EPET (Barnes and Walker, 1981 ; West et al., 
1986), and neurite outgrowth is inhibited by ethanol exposure in culture (Heaton et al., 
1993; Saunders et al., 1997), it is possible that ethanol may specifically disrupt the latter 
pathway of the trk intracellular cascade. 



155 

Future research will determine where the cascade might be affected. Each protein in 
the two trkC pathways should be examined for phosphorylation using antibodies for 
phosphotyrosine by western blot. Proteins would be obtained by immunoprecipitation of 
cell lysates. The time course of the reactions following neurotrophin binding to trk is 
relatively short. For example, SHC (a protem in the trkC intracellular cascade with a src 
homology domain) is phosphorylated just one minute after neurotrophin is introduced to 
the culture medium. Therefore, hippocampal cultures established to study this 
phenomenon do not have to be established for a long period before the analysis begins 
(Stephens et al., 1994). These future experiments should allow us to further define any 
interaction of ethanol and trk receptors. 

The results of this doctoral research do suggest some new ideas about the nature of 
ethanol neurotoxicity. Smce most of the genes studied m Chapter 4 were not affected 
significantly by CPET or EPET, ethanol does have the ability to alter the expression of 
discrete genes. This suggests that ethanol alters the activity of specific enhancers or 
repressors in the nucleus. Perhaps this result is achieved through changes in methylation 
of fetal DNA. Above, it was mentioned that methylation serves to prevent enhancers or 
repressors from binding to DNA and may alter gene expression (Holliday, 1987) and that 
ethanol is known to alter methylation of fetal DNA (Garro et al., 1991). By interfering 
with methylation at a specific site in the genome, ethanol may act in ways that are more 
specific than had previously been thought. 

The aim of all FAS research is to answer the question: "How does ethanol cause its 
toxic effects in the nervous system?" By comparing this research to other studies, some 
hypotheses can be made. For example, ethanol is now known to affect neurotrophin 
receptor gene expression and alter neurotrophic activity in the hippocampus (Heaton et al., 
1995c). NTFs are known to produce a variety of effects in the hippocampus. For 
example, BDNF and NGF have been shown to regulate a number of peptides in the rat 
hippocampus (Croll et al., 1994). Specifically, BDNF increases NT-3 activity in the 



156 

hippocampus (Lindholm et al., 1994) while BDNF, NGF, and NT-3 have been shown to 
induce choline acetyltransf erase (ChAT) activity in the septohippocampal neurons 
(Alderson et al., 1990; Auberger et al., 1987; Gnahn et al., 1983; Nonner et al, 1996). 
Prenatal ethanol exposure has been shown to slightly reduce ChAT activity in the sepaim at 
P7, while sparing septal neurons from any significant cell death (Swanson et al., 1995; 
Swanson et al., 1996). The reduction of ChAT activity was transient in nature and did not 
extend to any later ages. In light of the present results, the change in ChAT activity could 
be due to the reduction in trkC gene expression if trkC is also reduced in the septum 
following ethanol exposure. If less receptor is available for NT-3 to bind, less signal from 
trkC will be produced intracellularly. Therefore, less of an effect (in this case ChAT 
activity) would result in ethanol exposed animals. Future studies should attempt to 
determine if such a relationship does indeed exist. 

As was stated above, the results of Chapter 3 are especially important because they 
suggest that ethanol can increase motoneuron number during embryonic ethanol exposure. 
The implication of these experiments may be a drug treatment for FAS. One issue that 
inevitably arises when discussing this subject is delivery of the drug to the population of 
interest. The main difficulty is that most NTFs cannot cross the blood brain barrier 
(Anderson et al., 1995). Therefore, an NTF must be delivered by direct administration to 
the brain (which is invasive) or by some other vector. The use of viral vectors is not 
invasive, but the immune system will eventually hinder the process. Other diseases have 
been treated with NTFs include Parkinson's Disease and amyotrophic lateral sclerosis. 
Specific NTFs that have been used as potential therapies for Parkinson's disease include 
GDNF(Lapchaketal., 1997) and lGF-1 (Festoff et al, 1995). Amyotrophic lateral 
sclerosis has been treated with GDNF (Gimenez y Ribotta et al., 1997), BDNF (Gimenez y 
Ribotta et al., 1997), and CNTF (Aebischer et al., 1996; Stambler et al., 1998). While 
exhibiting success in animal models of the disease, these therapies have not yet provided a 
cure for human subjects. Beyond defining which NTFs are successful in preventing 



157 

ethanol toxicity, future research should focus on delivering this protection to mammalian 
and human test subjects. This goal is important because the ami for conductmg this 
research is to treat this harmful disorder in humans when prevention has failed. This next 
step-developing a therapy or cure-is the only way that research in this field will ultimately 
be judged. 



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BIOGRAPHICAL SKETCH 
Douglas M. Bradley was bom in Birmingham, Alabama, in 1971. He lived there 
until 1990 when he entered the University of Florida as a freshman. There he met his wife- 
to-be, Korey Rothman, and majored in Neurobiological Sciences. Among the highlights of 
his undergraduate days were a year in the Pride of the Sunshine Marching Band, induction 
in Phi Beta Kappa, 1st place in the Undergraduate Research Symposium, and highest 
honors upon graduation. In the Fall of 1994, he began his tenure in the Department of 
Neuroscience. The achievements for which he is most proud include a National Science 
Foundation Predoctoral Fellowship, Grinter Predoctoral Fellowship, and his graduation 
within four years of beginning this program. 



178 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 



Marieta B. Heaton, Chair 
Professor of Neuroscience 

I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 

A. JohlfMacLennan 

Associate Professor of Neuroscience 

I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 



Kathleen Shiverick 

Professor of Pharmacology and Therapeutics 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in SQOpe and quality, as a 
dissertation for the degree of Doctor of Philosophy./ \ |' i 




Wolfgang J. Streit 

Associate Professor of Neuroscience 



I certify that I have read this study and that in my opinion it conforms to acceptable 
standards of scholarly presentation and is fully adequate, in scope and quality, as a 
dissertation for the degree of Doctor of Philosophy. 



Don W. Walker 
Professor of Neuroscience 

This dissertation was submitted to the Graduate Faculty of the College of Medicine 
and to the Graduate School and was accepted asp^iayu}l|illment of the requirements for 
the degree of Doctor of Philosophy. 

August 1998 




2ge of Medicine 
Dean, Graduate School